Use of anp32 protein in maintaining the polymerase activity of influenza virus in hosts

ABSTRACT

The present invention provides a recombinant sequence information of a key host factor ANP32A/B which is necessary for the replication of influenza virus in a host. More specifically, the present invention relates to a 129-130 motif and a 149 site of the host factor ANP32A/B protein, which are key active sites for exerting its ability to promote the replication of influenza virus, and are also potential targeting sites of anti-influenza drugs.

TECHNICAL FIELD

The present invention relates to key host factors necessary forinfluenza virus replication in a host. More specifically, it relates tothe key host factors ANP32A and ANP32B necessary for influenza virusreplication in hosts.

BACKGROUND

Influenza is one of the most serious human infectious diseases caused byinfluenza virus. A global influenza epidemic will infect about 20%-40%of the population (Basler C F, et al. Sequence of the 1918 pandemicinfluenza virus nonstructural gene (NS) segment and characterization ofrecombinant viruses bearing the 1918 NS genes[J]. Proc Natl Acad SciUSA, 2001, 98(5):2746-2751.), and will pose a huge threat to human life,health and social life. Influenza virus is an enveloped RNA virusbelonging to the family of orthomyxoviridae, and is a spherical orpolymorphic particle with a diameter of 80-120 nm. Influenza viruses canbe classified into type A, B, C, and D, wherein influenza A virus is themain virus responsible for seasonal influenza and historical influenzapandemics due to its rapid mutation and evolution, variable antigens,strong infectivity and pathogenicity, and rapid spread (Hardelid P, al.Excess mortality monitoring in England and Wales during the influenzaA(H1N1) 2009 pandemic[J]. Epidemiol Infect, 2011, 139(9):1431-1439; YangL, et al. Excess mortality associated with the 2009 pandemic ofinfluenza A(H1N1) in Hong Kong[J]. Epidemiol Infect, 2012,140(9):1542-1550). The influenza A virus caused four global influenzapandemics in 1918, 1957, 1968, and 2009. The rapid and widespread spreadof the virus in the population may be related to its increasedadaptability and replication capacity in the host, while the specificand efficient replication in the host and its adaptability to the hostare mainly determined by RNA-dependent RNA polymerases (Eisfeld A J, etal. At the centre: influenza A virus ribonucleoproteins. [J]. Nat RevMicrobiol. 2015 January; 13(1):28-41.).

The genome of influenza virus contains eight fragments ofnegative-strand RNA, encoding a total of 11 proteins, wherein the RNApolymerase is a heteromultimer consisting of PB1, PB2 and PA encoded bythe first three sequences (Area E, et al. 3D structure of the influenzavirus polymerase complex: localization of subunit domains[J]. Proc NatlAcad Sci USA, 2004, 101(1):308-313; Eisfeld A J, et al. At the centre:influenza A virus ribonucleoproteins. [J]. Nat Rev Microbiol. 2015January; 13(1):28-41). RNA polymerase is the material basis for thetranscription and replication of influenza virus in host cells, and hasan important influence on the pathogenicity and host range of the virus(Neumann G, et al. Host range restriction and pathogenicity in thecontext of influenza pandemic [J]. Emerg Infect Dis, 2006,12(6):881-886.). Various factors that interact with influenza viruspolymerase and subunits thereof in the host cell, such as RNA helicaseDDX, importin a, etc., have influence on the assembly and activity ofinfluenza virus polymerase trimers (Bortz E, et al. Host- andstrain-specific regulation of influenza virus polymerase activity byinteracting cellular proteins[J]. MBio, 2011, 2(4); Gabriel G, et al.Differential use of importin-alpha isoforms governs cell tropism andhost adaptation of influenza virus[J]. Nat Commun, 2011, 2:156.).Therefore, the interaction of influenza virus polymerase with hostproteins is an important factor in determining the virulence and hostrange of the virus.

The cross-species spread of influenza virus and its host restrictionmechanism have always been the focus of research in the field. The hostprotein ANP32 (acidic nuclear phosphoprotein) is thought to be involvedin the synthesis of viral RNA in infected cells. Previous studies havefocused on the role of the protein in cellular physiological activitiessuch as intracellular transport, cell death pathways, and regulation oftranscription, etc., as well as the correlation of ANP32 protein withtumors and nervous system diseases. The members of ANP32 protein familyare numerous and structurally similar, consisting of a sphericalamino-terminus rich in leucine repeats (LRRs) and an extended carboxylterminus rich in acidic amino acids (LCAR) (Reilly P T, et al. Crackingthe ANP32 whips: important functions, unequal requirement, and hints atdisease implications[J]. Bioessays, 2014, 36(11):1062-1071.). Theprotein family is found in animals, plants and protista, but not foundin yeast and other fungi, which indicates that the protein familyoriginates from eukaryotes and is lost in fungi due to certain reasons.It has been reported that this family has 8 members in humans, 3 ofwhich are more conservative in vertebrates, including ANP32A, ANP32B andANP32E, and the most widely studied are ANP32A and ANP32B. The aminoacid sequence homology of ANP32A and ANP32B protein is 70%. Theexistence of the LRRs region at the amino terminal of the ANP32 proteinmakes the protein hydrophobic, which helps ANP32 protein to bind toother proteins and play different biological roles; the carboxylterminal is rich in acidic amino acids and contains a nuclearlocalization signal, KRKR, which makes the protein capable ofinteracting with the basic proteins in the nucleus and shuttling in thekaryoplasm (Matsubae, Masami, et al. “Characterization of the nucleartransport of a novel leucine-rich acidic nuclear protein-like protein.”Febs Letters 468.2-3(2000):171-175; Matsuoka, K., et al. “A NuclearFactor Containing the Leucine-Rich Repeats Expressed in MurineCerebellar Neurons.” Proceedings of the National Academy of Sciences ofthe United States of America 91.21(1994):9670.). The protein can also beexpressed on the cell surface and even secreted outside of the cell.These characteristics allow the ANP32 protein to participate in avariety of biological processes in cytoplasm and nucleus. It wasreported in the papers that ANP32A and ANP32B function astranscriptional regulators in the nucleus: {circle around (1)} as acomponent of the inhibitor of the histone acetyltransferase (INHAT)complex, involved in regulating transcription (Kadota, S, et al. “pp32,an INHAT component, is a transcription machinery recruiter for maximalinduction of IFN-stimulated genes.” Journal of Cell Science 124.Pt6(2011):892.); {circle around (2)} as the ligand of mRNA binding proteinHuR and nuclear export factor CRM1, may accelerate the nuclear export ofmRNA chain rich in adenosine (Brennan C M, et al. Protein Ligands to HurModulate Its Interaction with Target Mrnas in Vivo[J]. Journal of CellBiology, 2000, 151(1):1.), and this function can control both the mRNAof the cell host (Fries, Barbara, et al. “Analysis of NucleocytoplasmicTrafficking of the HuR Ligand APRIL and Its Influence on CD83Expression.” Journal of Biological Chemistry 282.7(2007):4504-15.), andthe mRNA of the virus (Bodem, J, et al. “Foamy virus nuclear RNA exportis distinct from that of other retroviruses.” Journal of Virology85.5(2011):2333-2341.). In addition, ANP32 protein can also play animportant role in the cytoplasm, for example, ANP32A can bind tomicrotubule-associated proteins (Ulitzur, N, M, et al. “Mapmodulin: apossible modulator of the interaction of microtubule-associated proteinswith microtubules.” Proceedings of the National Academy of Sciences ofthe United States of America 94.10(1997):5084.), which affects thetransport and signal transmission of intracellular materials, maintainsthe cell morphology and the spatial distribution of organelles, etc.;ANP32 protein can activate apoptotic bodies, and regulate the cell deathpathways (Pan, Wei, et al. “PHAPI/pp32 Suppresses Tumorigenesis byStimulating Apoptosis.” Journal of Biological Chemistry284.11(2009):6946.).

After Shapira screened the host protein ANP32A through the yeasttwo-hybrid method in 2009 and initially discovered that it wasassociated with influenza virus infection, scientists have successivelyconducted researches on the interaction between this protein family andinfluenza virus. In 2011, Bradel-Tretheway et al. discovered that ANP32Aand ANP32B bond to influenza virus polymerase through proteomics; in2014, Watanabe et al. demonstrated that ANP32A and ANP32B can affectvRNA synthesis after the influenza virus infected cells through RANilibrary screening; in 2015, Sugiyama et al. found that ANP32A and ANP32Bcan promote the synthesis of cRNA-vRNA in influenza virus replication,and demonstrated that ANP32 protein interacted with influenza viruspolymerase trisubunit (PB2/PB1/PA) polymer through biological techniquessuch as CO-IP, and had nothing to do with NP protein (Sugiyama K, et al.pp32 and APRIL are host cell-derived regulators of influenza virus RNAsynthesis from cRNA[J]. Elife, 2015, 4; Watanabe T, et al. Influenzavirus-host interactome screen as a platform for antiviral drugdevelopment[J]. Cell Host Microbe, 2014, 16(6):795-805.). Subsequently,the British scholar Wendy S. Barclay's research team revealed for thefirst time that the activity of avian influenza virus RNA polymerase inmammalian cells is related to the species-specificity of ANP32A (Long JS, et al. Species difference in ANP32A underlies influenza A viruspolymerase host restriction[J]. Nature, 2016, 529(7584):101-104.). Studyfound that compared to mammals, avian-derived ANP32A contains a sequenceof 33 amino acid inserted between the LRRs and LCAR region. Thischaracteristic of sequence determines the characteristic thatavian-derived ANP32A specifically activates the polymerase activity ofavian-derived influenza virus. Avian-derived influenza virus (H5N1/H7N9)can make the polymerase more adaptive to mammalian ANP32A only afterobtaining mutations in PB2 E627K. Therefore, it is inferred that ANP32Ais an important host protein that supports influenza virus replication,but the specific molecular mechanism of its interaction with polymeraseremains to be further explored.

The harm of seasonal influenza and global pandemic influenza hasattracted widespread attention from the society. At present, preliminarysuccess has been made on anti-influenza virus drugs. However, due to theclinical widespread use of existing drugs, influenza viruses areconstantly mutating, and have developed different degrees of resistanceto these drugs, which makes development and screening of newanti-influenza virus drugs increasingly important. The present inventiondemonstrates that ANP32A and ANP32B are common host proteins used byinfluenza viruses in cells of different species of animals, and thatboth or either of the two proteins are necessary for the replication ofinfluenza viruses in the host. Furthermore, the present invention alsofinds the key amino acid positions in the two proteins, and the mutationof the key amino acids makes the influenza virus almost impossible toreplicate. This discovery can provide a direct basis for the furtherdesign of anti-influenza drugs or anti-influenza animals.

SUMMARY OF THE INVENTION

In one aspect of the invention, it relates to a mutated ANP32 proteinhaving one or more mutations selected from the group consisting of:

-   -   the amino acid at position 129 is substituted with isoleucine I,        lysine K, aspartic acid D, valine V, proline P, tryptophan W,        histidine H, arginine R, glutamine Q, glycine G, or glutamic        acid E,    -   the amino acid at position 130 is substituted with asparagine N,        phenylalanine F, lysine K, leucine L, valine V, proline P,        isoleucine I, methionine M, tryptophan \V, histidine H, arginine        R, glutamine Q, or tyrosine Y,    -   the amino acid at position 149 is substituted with alanine A,    -   the amino acid at position 151 is substituted with alanine A,        and    -   the amino acids at positions 60 and 63, positions 87, 90, 93 and        95, positions 112, 115 and 118 are substituted with alanine,    -   when the ANP32 protein is chicken ANP32B protein, duck ANP32B        protein or turkey ANP32B protein, the amino acid at position 129        is not isoleucine I and the amino acid at position 130 is not        asparagine N,    -   when the ANP32 protein is murine ANP32A, the amino acid at        position 130 is not alanine A,    -   wherein, when the ANP32 protein is an ANP32A protein, the amino        acid positions correspond to the amino acid positions of a        chicken ANP32A protein of GenBank No. XP_413932.3;    -   when the ANP32 protein is an ANP32B protein, the amino acid        positions correspond to the amino acid positions of a human        ANP32B protein of GenBank No. NP 006392.1,    -   wherein, preferably, the amino acids at positions 87, 90, 93 and        95 are from the mammalian ANP32B protein.

In one aspect of the invention, it relates to a mutated ANP32 protein,wherein one or more of the amino acid segments selected from the groupconsisting of the following are deleted or substituted with alanine:amino acids at positions 61-70, amino acids at positions 71-80, aminoacids at positions 81-90, amino acids at positions 91-100, amino acidsat positions 101-110, amino acids at positions 111-120, amino acids atpositions 121-130, amino acids at positions 131-140, amino acids atpositions 141-150, amino acids at positions 151-160,

-   -   wherein, when the ANP32 protein is an ANP32A protein, the amino        acid positions correspond to the amino acid positions of a        chicken ANP32A protein of GenBank No. XP_413932.3;

when the ANP32 protein is an ANP32B protein, the amino acid positionscorrespond to the amino acid positions of a human ANP32B protein ofGenBank No. NP 006392.1.

In one aspect of the invention, it relates to a mutated ANP32 protein,wherein when the ANP32 protein is a human ANP32B protein, one or more ofthe amino acid segments selected from the group consisting of thefollowing are deleted or substituted with alanine: amino acids atpositions 21-30, amino acids at positions 41-50, amino acids atpositions 51-60 or amino acids at positions 161-170, and the amino acidpositions correspond to the amino acid positions of the human ANP32Bprotein of GenBank No. NP 006392.1.

In one aspect of the invention, it relates to a mutated ANP32 protein,wherein when the ANP32 protein is a chicken ANP32B protein, one or moreof the amino acid segments selected from the group consisting of thefollowing are deleted or substituted with alanine: amino acids atpositions 161-170, amino acids at positions 171-180 or amino acids atpositions 191-200, and the amino acid positions correspond to the aminoacid positions of the chicken ANP32B protein of GenBank No. XP_413932.3.

In an embodiment of the present invention, the ANP32 protein is selectedfrom ANP32A or ANP32B, preferably derived from chicken, human, zebrafinch, duck, turkey, pig, mouse or horse, more preferably derived fromchicken or human, most preferably is human ANP32B, or chicken ANP32A

In one aspect of the present invention, it relates to a use of ANP32protein in maintaining the polymerase activity of influenza virus.Preferably, the ANP32 protein is an avian-derived ANP32 protein, and theinfluenza virus is selected from avian-derived or mammal-derivedinfluenza virus; alternatively, the ANP32 protein is a mammal-derivedANP32 protein, and the influenza virus is a mammal-derived influenzavirus; preferably, the influenza virus is selected from a human, canine,avian, or equine influenza virus, and preferably, the ANP32 protein isselected from ANP32A protein and ANP32B protein, more preferably, theANP32 protein is derived from chicken, human, zebra finch, duck, turkey,pig, mouse or horse, and preferably, the ANP32 protein is not achicken-derived ANP32B or mouse-derived ANP32A.

In one aspect of the present invention, it relates to a use of ANP32protein in reducing the polymerase activity of influenza virus.Preferably, the influenza virus is selected from human, canine, avian,or equine influenza virus; preferably, the ANP32 protein is selectedfrom ANP32A protein and ANP32B protein, more preferably, the ANP32protein is derived from chicken, human, zebra finch, duck, turkey, pig,mouse or horse, and more preferably, the ANP32 protein is a mutatedANP32 protein as defined in any one of claim 1-4, or ANP32 protein is achicken-derived ANP32B protein or mouse-derived ANP32A protein.

In one aspect of the invention, it relates to a method of reducing thepolymerase activity of influenza virus, including subjecting the ANP32protein to one or more mutations selected from the group consisting of:

-   -   the amino acid at position 129 is substituted with isoleucine I,        lysine K, aspartic acid D, valine V, proline P, tryptophan W,        histidine H, arginine R, glutamine Q, glycine G, or glutamic        acid E,    -   the amino acid at position 130 is substituted with asparagine N,        phenylalanine F, lysine K, leucine L, valine V, proline P,        isoleucine I, methionine M, tryptophan W, histidine H, arginine        R, glutamine Q, or tyrosine Y,    -   the amino acid at position 149 is substituted with alanine A,    -   the amino acid at position 151 is substituted with alanine A,        and    -   the amino acids at positions 60 and 63, positions 87, 90, 93 and        95, positions 112, 115 and 118 are substituted with alanine,

when the ANP32 protein is a chicken ANP32B protein, duck ANP32B proteinor turkey ANP32B protein, the amino acid at position 129 is notisoleucine and the amino acid at position 130 is not asparagine N,

when the ANP32 protein is murine ANP32A, the amino acid at position 130is not alanine A,

wherein, when the ANP32 protein is an ANP32A protein, the amino acidpositions correspond to the amino acid positions of a chicken ANP32Aprotein of GenBank No. XP_413932.3;

when the ANP32 protein is an ANP32B protein, the amino acid positionscorrespond to the amino acid positions of a human ANP32B protein ofGenBank No. NP 006392.1,

wherein, preferably, the amino acids at positions 87, 90, 93 and 95 arefrom the mammalian ANP32B protein.

In an embodiment of the present invention, wherein the polymeraseactivity of influenza virus is lost,

wherein the ANP32 protein is subjected to one or more mutations selectedfrom the group consisting of:

the amino acid at position 129 is substituted with isoleucine I, lysineK or aspartic acid D,

the amino acid at position 130 is substituted with asparagine N,phenylalanine F or lysine K,

the amino acids at positions 87, 90, 93 and 95, positions 112, 115 and118 are substituted with alanine.

In one aspect of the invention, it relates to a method of reducing thepolymerase activity of influenza virus, including one or more of theamino acid segments of ANP32 protein selected from the group consistingof: amino acids at positions 61-70, amino acids at positions 71-80,amino acids at positions 81-90, amino acids at positions 91-100, aminoacids at positions 101-110, amino acids at positions 111-120, aminoacids at positions 121-130, amino acids at positions 131-140, aminoacids at positions 141-150, amino acids at positions 151-160, aredeleted or substituted with alanine,

-   -   wherein, when the ANP32 protein is an ANP32A protein, the amino        acid positions correspond to the amino acid positions of a        chicken ANP32A protein of GenBank No. XP_413932.3;

when the ANP32 protein is an ANP32B protein, the amino acid positionscorrespond to the amino acid positions of a human ANP32B protein ofGenBank No. NP 006392.1.

In an embodiment of the present invention, wherein the polymeraseactivity of influenza virus is lost,

wherein one or more of the amino acid segments of ANP32 protein selectedfrom the group consisting of: amino acids at positions 71-80, aminoacids at positions 81-90, amino acids at positions 91-100, amino acidsat positions 101-110, amino acids at positions 111-120, amino acids atpositions 121-130, amino acids at positions 131-140, amino acids atpositions 141-150, amino acids at positions 151-160, are deleted orsubstituted with alanine.

In one aspect of the invention, it relates to a method of reducing thepolymerase activity of influenza virus, including deleting orsubstituting one or more of the amino acid segments of human ANP32Bprotein selected from the group consisting of: amino acids at positions21-30, amino acids at positions 41-50, amino acids at positions 51-60 oramino acids at positions 161-170 with alanine, the amino acid positionscorrespond to the amino acid positions of the human ANP32B protein ofGenBank No. NP_006392.1.

In one aspect of the invention, it relates to a method of reducing thepolymerase activity of influenza virus, including deleting orsubstituting one or more of the amino acid segments of chicken ANP32Aprotein selected from the group consisting of: amino acids at positions161-170, amino acids at positions 171-180 or amino acids at positions191-200 with alanine, the amino acid positions correspond to the aminoacid positions of the chicken ANP32A protein of GenBank No. XP_413932.3.

In an embodiment of the present invention, wherein the polymeraseactivity of influenza virus is lost,

wherein, one or more of the amino acid segments of chicken ANP32Aprotein selected from the group consisting of: amino acids at positions161-170, amino acids at positions 171-180 are deleted or substitutedwith alanine.

In an embodiment of the present invention, wherein the ANP32 protein isselected from ANP32A or ANP32B, preferably derived from chicken, human,zebra finch, duck, turkey, pig, mouse or horse, and more preferablyderived from chicken or human. Most preferably, it is a human ANP32B, orchicken ANP32A; preferably, the influenza virus is selected from human,canine, avian or equine influenza virus.

In one aspect of the present invention, it relates to a use of one ormore of the amino acid segments of ANP32 protein in maintaining thepolymerase activity of influenza virus, wherein the amino acid segmentis selected from the group consisting of:

amino acids at positions 61-70, amino acids at positions 71-80, aminoacids at positions 81-90, amino acids at positions 91-100, amino acidsat positions 101-110, amino acids at positions 111-120, amino acids atposition 121-130, amino acids at positions 131-140, amino acids atpositions 141-150, amino acid at positions 151-160, amino acids atpositions 161-170 of chicken ANP32A protein, amino acids at positions171-180 of chicken ANP32A protein, amino acids at positions 191-200 ofchicken ANP32A protein, amino acids at positions 21-30 of human ANP32Bprotein, amino acids at positions 41-50 of human ANP32B protein, aminoacids at positions 51-60 of human ANP32B protein or amino acids atpositions 161-170 of human ANP32B protein,

-   -   wherein, when the ANP32 protein is an ANP32A protein, the amino        acid positions correspond to the amino acid positions of a        chicken ANP32A protein of GenBank No. XP_413932.3;    -   when the ANP32 protein is an ANP32B protein, the amino acid        positions correspond to the amino acid positions of a human        ANP32B protein of GenBank No. NP_006392.1,        preferably, the ANP32 protein is an avian-derived ANP32 protein,        and the influenza virus is selected from an avian-derived or        mammal-derived influenza virus;

alternatively, the ANP32 protein is a mammal-derived ANP32 protein, andthe influenza virus is a mammal-derived influenza virus.

In one aspect of the present invention, it relates to the use of one ormore of the amino acid segments of ANP32 protein in reducing theactivity of influenza virus polymerase, wherein the amino acid segmentis selected from the group consisting of:

amino acids at positions 61-70, amino acids at positions 71-80, aminoacids at positions 81-90, amino acids at positions 91-100, amino acidsat positions 101-110, amino acids at positions 111-120, amino acids atposition 121-130, amino acids at positions 131-140, amino acids atpositions 141-150, amino acid at positions 151-160, amino acids atpositions 161-170 of chicken ANP32A protein, amino acids at positions171-180 of chicken ANP32A protein, amino acids at positions 191-200 ofchicken ANP32A protein, amino acids at positions 21-30 of human ANP32Bprotein, amino acids at positions 41-50 of human ANP32B protein, aminoacids at positions 51-60 of human ANP32B protein or amino acids atpositions 161-170 of human ANP32B protein,

-   -   wherein, when the ANP32 protein is an ANP32A protein, the amino        acid positions correspond to the amino acid positions of a        chicken ANP32A protein of GenBank No. XP_413932.3;    -   when the ANP32 protein is an ANP32B protein, the amino acid        positions correspond to the amino acid positions of a human        ANP32B protein of GenBank No. NP_006392.1,

preferably, wherein the ANP32 protein is selected from ANP32A or ANP32B,preferably derived from chicken, human, zebra finch, duck, turkey, pig,mouse or horse, more preferably derived from chicken or human, mostpreferably is human ANP32B or chicken ANP32A; preferably, the influenzavirus is selected from human, canine, avian or equine influenza virus.

In one aspect of the present invention, it relates to a kit comprisingat least one reagent or a set of reagents, wherein the at least onereagent or set of reagents is used to determine the type of amino acidat one or more positions of an ANP32 protein selected from the groupconsisting of: amino acid at position 129, amino acid at position 130,amino acid at position 149, amino acid at position 151, amino acid atposition 60, amino acid at position 63, amino acid at position 87, aminoacid at position 90, amino acid at position 93, amino acid at position95, amino acid at position 112, amino acid at position 115, or aminoacid at position 118, wherein

when the amino acid at position 129 is isoleucine I, lysine K, asparticacid D, valine V, proline P, tryptophan W, histidine H, arginine R,glutamine Q, glycine G, or glutamic acid E,

the amino acid at position 130 is asparagine N, phenylalanine F, lysineK, leucine L, valine V, proline P, isoleucine I, methionine M,tryptophan W, histidine H, arginine R, glutamine Q, or tyrosine Y,

the amino acid at position 149 is alanine A,

the amino acid at position 151 is alanine A,

the amino acids at positions 60 and 63, at positions 87, 90, 93 and 95,at positions 112, 115, and 118 are alanine, the ability of the ANP32protein to support the activity of influenza polymerase is decreased,

-   -   wherein, when the ANP32 protein is an ANP32A protein, the amino        acid positions correspond to the amino acid positions of a        chicken ANP32A protein of GenBank No. XP_413932.3;    -   when the ANP32 protein is an ANP32B protein, the amino acid        positions correspond to the amino acid positions of a human        ANP32B protein of GenBank No. NP_006392.1, wherein, preferably,        the amino acids at positions 87, 90, 93 and 95 are from the        mammalian ANP32B protein.

In one aspect of the present invention, it relates to an oligonucleotideprimer for determining the type of amino acid of ANP32 protein selectedfrom the group consisting of: amino acid at position 129, amino acid atposition 130, amino acid at position 149, amino acid at position 151,amino acid at position 60, amino acid at position 63, amino acid atposition 87, amino acid at position 90, amino acid at position 93, aminoacid at position 95, amino acid at position 112, amino acid at position115, and amino acid at position 118, wherein

when the amino acid at position 129 is isoleucine I, lysine K, asparticacid D, valine V, proline P, tryptophan W, histidine H, arginine R,glutamine Q, glycine G, or glutamic acid E,

the amino acid at position 130 is asparagine N, phenylalanine F, lysineK, leucine L, valine V, proline P, isoleucine I, methionine M,tryptophan W, histidine H, arginine R, glutamine Q, or tyrosine Y,

the amino acid at position 149 is alanine A,

the amino acid at position 151 is alanine A,

the amino acids at positions 60, 63, or the amino acids at positions 87,90, 93 and 95, or the amino acids at positions 112, 115 and 118 arealanine,

the ability of ANP32 protein to support the activity of influenzapolymerase is decreased,

-   -   wherein, when the ANP32 protein is an ANP32A protein, the amino        acid positions correspond to the amino acid positions of a        chicken ANP32A protein of GenBank No. XP_413932.3;

when the ANP32 protein is an ANP32B protein, the amino acid positionscorrespond to the amino acid positions of a human ANP32B protein ofGenBank No. NP_006392.1,

wherein, preferably, the amino acids at positions 87, 90, 93 and 95 arefrom the mammalian ANP32B protein.

In an embodiment of the present invention, the oligonucleotide primer ispreferably at least 20 bases in length. For example at least 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 bases in length.

In an embodiment of the present invention, the oligonucleotide sequenceis selected from SEQ ID NOs: 155-156, 163-166, 167-256, and 375-380.

In an embodiment of the present invention, the ANP32 protein is selectedfrom ANP32A or ANP32B, preferably derived from chicken, human, zebrafinch, duck, turkey, pig, mouse or horse, more preferably derived fromchicken or human. Most preferably, the ANP32 protein is human ANP32B, orchicken ANP32A.

In one aspect of the present invention, it relates to a method ofproducing an animal, including the step of subjecting the ANP32 proteinin the animal to one or more mutations selected from the groupconsisting of:

-   -   the amino acid at position 129 is substituted with isoleucine I,        lysine K, aspartic acid D, valine V, proline P, tryptophan W,        histidine H, arginine R, glutamine Q, glycine G, or glutamic        acid E,    -   the amino acid at position 130 is substituted with asparagine N,        phenylalanine F, lysine K, leucine L, valine V, proline P,        isoleucine I, methionine M, tryptophan \V, histidine H, arginine        R, glutamine Q, or tyrosine Y,    -   the amino acid at position 149 is substituted with alanine A,    -   the amino acid at position 151 is substituted with alanine A,        and    -   the amino acids at positions 60 and 63, positions 87, 90, 93 and        95, positions 112, 115 and 118 are substituted with alanine,    -   the amino acids at positions 61-70, amino acids at positions        71-80, amino acids at positions 81-90, amino acids at positions        91-100, amino acids at positions 101-110, amino acids at        positions 111-120, amino acid at position 121-130, amino acids        at positions 131-140, amino acids at positions 141-150, amino        acids at positions 151-160, amino acids at positions 161-170 of        chicken ANP32A protein, amino acids at positions 171-180 of        chicken ANP32A protein, amino acids at positions 191-200 of        chicken ANP32A protein, amino acids at positions 21-30 of human        ANP32B protein, amino acids at positions 41-50 of human ANP32B        protein, amino acids at positions 51-60 of human ANP32B protein        or amino acids at positions 161-170 of human ANP32B protein are        substituted with alanine,    -   wherein, when the ANP32 protein is chicken ANP32B protein, duck        ANP32B protein or turkey ANP32B protein, the amino acid at        position 129 is not isoleucine I and the amino acid at position        130 is not asparagine N,    -   wherein, when the ANP32 protein is murine ANP32A, the amino acid        at position 130 is not alanine A,    -   wherein, when the ANP32 protein is an ANP32A protein, the amino        acid positions correspond to the amino acid positions of a        chicken ANP32A protein of GenBank No. XP_413932.3;    -   when the ANP32 protein is an ANP32B protein, the amino acid        positions correspond to the amino acid positions of a human        ANP32B protein of GenBank No. NP_006392.1,

wherein, preferably, the amino acids at positions 87, 90, 93 and 95 arefrom the mammalian ANP32B protein.

In an embodiment of the present invention, the animal is selected fromchicken, human, zebra finch, duck, turkey, pig, mouse or horse.

In one aspect of the present invention, it relates to the use of ANP32protein or amino acid(s) thereof as a target in preparing a medicamentfor treating a disease caused by an influenza virus infection, whereinthe amino acid(s) is (are) selected from the amino acid(s) located atthe following positions or segments,

wherein the amino acid(s) is (are) selected from one or more of thefollowing amino acids: amino acid at position 129, amino acid atposition 130, amino acid at position 149, amino acid at position 150,amino acids at positions 60 and 63, amino acids at positions 87, 90, 93and 95, amino acids at positions 112, 115 and 118, amino acids atpositions 61-70, amino acids at positions 71-80, amino acids atpositions 81-90, amino acids at positions 91-100, amino acids atpositions 101-110, amino acids at positions 111-120, amino acids atposition 121-130, amino acids at positions 131-140, amino acids atpositions 141-150, amino acids at positions 151-160, amino acid atpositions 161-170 of chicken ANP32A protein, amino acids at positions171-180 of chicken ANP32A protein, amino acids at positions 191-200 ofchicken ANP32A protein, amino acids at positions 21-30 of human ANP32Bprotein, amino acids at positions 41-50 of human ANP32B protein, aminoacids at positions 51-60 of human ANP32B protein or amino acids atpositions 161-170 of human ANP32B protein, preferably, the amino acidsat positions 87, 90, 93 and 95 are from the ANP32B protein of mammalPreferably, the ANP32 protein is ANP32A or ANP32B protein, preferablyderived from chicken, human, zebra finch, duck, turkey, pig, mouse orhorse; preferably, the influenza virus is selected from human, canine,avian or equine influenza virus.

In one aspect of the present invention, it relates a method of screeningfor a candidate drug for treating an influenza virus infection,including the following steps:

(1) knocking out the ANP32A and/or ANP32B protein from a cell linecontaining ANP32A and/or ANP32B protein, to obtain a cell line in whichANP32A protein and ANP32B protein are knocked out,

(2) transfecting the knockout cell line obtained in step (1) with aplasmid encoding ANP32A and/or ANP32B protein and a plasmid encodinginfluenza virus polymerase,

(3) contacting the knockout cell line with a candidate, wherein thecontacting can be performed simultaneously with or separately from thetransfection of step (2),

wherein that the cell line treated in step (3) does not expressinfluenza virus polymerase or has reduced expression of influenza viruspolymerase compared to a cell line containing ANP32A and/or ANP32B,indicates that the candidate is a candidate drug for treating influenzavirus infection.

In one embodiment, wherein the ANP32A and/or ANP32B protein is derivedfrom chicken, human, zebra finch, duck, turkey, pig, mouse or horse;preferably, the influenza virus is selected from human, canine, avian orequine influenza virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the map of PCAGGS vector.

FIG. 2 shows the map of PCAGGS-huANP32A vector.

FIG. 3 shows the detection of expression of ANP32 protein from variousspecies.

FIG. 4 shows the detection of the knockout cell line by fluorescentquantitative PCR: A is the quantitative detection of huANP32A on eachcell line, and B is the quantitative detection of huANP32B on each cellline.

FIG. 5 shows the detection of endogenous ANP32 protein in 293T wild-typeand knockout cell lines.

FIG. 6 shows the detection of activity of H1N1_(SC09) polymerase on 293Twild-type and knockout cell lines.

FIG. 7 shows the detection of activity of different polymerases on 293Twild-type and knockout cell lines. FIG. 7A is the detection of activityof H7N9_(AH13) polymerase on 293T wild-type and knockout cell lines;FIG. 7B is the detection of activity of WSN polymerase on 293T wild-typeand knockout cell lines; FIG. 7C is the detection of activity ofH3N2_(G11) polymerase on 293T wild-type and knockout cell lines; FIG. 7Dis the detection of activity of H3N8_(JL89) polymerase on 293T wild-typeand knockout cell lines; FIG. 7E is the detection of activity ofH3N8_(XJ07) polymerase on 293T wild-type and knockout cell lines.

FIG. 8 shows the activity assay of H1N1_(SC09) polymerase on DKO cellline when supplemented with different doses of huANP32A or huANP32B.

FIG. 9 shows that the activity of H1N1_(SC09) polymerase (A) andH7N9_(AH13) polymerase (B) on DKO cells is inhibited when supplementedwith excessive huANP32A or huANP32B.

FIG. 10 shows the influence of huANP32A on the RNA synthesis ofH1N1_(SC09) influenza virus by fluorescent quantitative detection.

FIG. 11 shows the influence of ANP32 protein of different species on thereplication of different influenza viruses. FIG. 11A shows the influenceof ANP32 protein of different species on the replication of H1N1_(SC09)influenza virus; FIG. 11B shows the influence of ANP32 protein ofdifferent species on the replication of H7N9_(AH13) influenza virus;FIG. 11C shows the influence of ANP32 protein of different species onthe replication of H3N2_(GD11) influenza virus; FIG. 11D shows theinfluence of ANP32 protein of different species on the replication ofH3N8_(XJ07) influenza virus; FIG. 11E shows the influence of ANP32protein of different species on the replication of H3N8_(JL89) influenzavirus; FIG. 11F shows the influence of ANP32 protein of differentspecies on the replication of H9N2_(ZJ12) influenza virus; FIG. 11Gshows the influence of ANP32 protein of different species on thereplication of WSN influenza virus.

FIG. 12 shows the detection of H1N1_(SC09) influenza virus; FIG. 12Ashows the amount of virus from different cell lines transfected withH1N1_(SC09) influenza virus; FIG. 12B shows the influence ofsupplementation with ANP32 protein on the amount of H1N1_(SC09)influenza virus from DKO cells.

FIG. 13 shows the detection of WSN influenza virus; FIG. 13A shows thevirus titers of different cell lines transfected with WSN influenzavirus;

FIG. 13B shows the influence of supplementation with ANP32 protein onthe virus titer of WSN influenza virus from DKO cells.

FIG. 14 shows the adaptation of point mutation of PB2 gene of H7N9subtype influenza virus to ANP32.

FIG. 15 shows the ANP32B sequences alignment, truncation and fragmentinterchange; FIG. 15A is an alignment of ANP32B sequences;

FIG. 15B shows an ANP32B truncation strategy; FIG. 15C shows the firstround of interchanging huANP32B with chANP32B fragments; FIG. 15D showsthe second round of interchanging huANP32B with chANP32B fragments.

FIG. 16 shows the ANP32B point mutation; FIG. 16A is the sequencealignment of ANP32B 110-161 regions; FIG. 16B shows the influence of thepoint mutation of huANP32B on H1N1_(SC09) polymerase activity.

FIG. 17 shows the influence of single point mutation on position 129 or130 of huANP32B on H1N1_(SC09) polymerase activity.

FIG. 18 shows the influence of single point mutation of huANP32B onH7N9_(AH13) polymerase activity.

FIG. 19 shows the influence of point mutation at position 129 or 130 ofhuANP32A on H1N1_(SC09) polymerase activity.

FIG. 20 shows the influence of point mutation of huANP32A on H7N9_(AH13)polymerase activity.

FIG. 21 shows the influence of chANP32A point mutation and chANP32Bpoint mutation on H7N9_(AH13) polymerase activity.

FIG. 22 shows the influence of point mutant at position 129 of chANP32Aon H7N9_(ZJ13) polymerase activity.

FIG. 23 shows the influence of point mutant at position 129 of chANP32Aon H7N9_(AH13) polymerase activity.

FIG. 24 shows the influence of point mutant at position 129 of chANP32Aon the activity of WSN polymerase.

FIG. 25 shows the influence of point mutant at position 130 of chANP32Aon H7N9_(ZJ13) polymerase activity.

FIG. 26 shows the influence of point mutant at position 130 of chANP32Aon H7N9_(AH13) polymerase activity.

FIG. 27 shows the influence of point mutant at position 130 of chANP32Aon the activity of WSN polymerase.

FIG. 28 shows the influence of huANP32B truncated mutant on H7N9_(AH13)polymerase activity.

FIG. 29 shows the influence of chANP32A truncated mutant on H7N9_(ZJ13)polymerase activity.

FIG. 30 shows the influence of chANP32A point mutant on H7N9_(AH13)polymerase activity.

FIG. 31 shows the influence of huANP32B point mutant on H7N9_(AH13)polymerase activity.

FIG. 32 shows the identification and sequencing result of site-directedmutant cell line of amino acid at position 129/130 of huANP32A andhuANP32B.

FIG. 33 shows the protein detection result of site-directed mutant cellline of amino acid at position 129/130 of huANP32A and huANP32B.

FIG. 34 shows the detection of activity of H1N1_(SC09) polymerase ondifferent cell lines.

FIG. 35 shows the detection of activity of H7N9_(AH13) polymerase ondifferent cell lines.

FIG. 36 shows the alignment of amino acid sequences of avian-derivedANP32A proteins.

FIG. 37 shows the alignment of amino acid sequences betweenavian-derived ANP32B protein and huANP32B protein.

FIG. 38 shows the alignment of amino acid sequences between chickenANP32A protein and mammalian ANP32A protein.

FIG. 39 shows the alignment of amino acid sequences of mammalian ANP32Bprotein.

FIG. 40 shows the detection of murine ANP32B protein expression.

FIG. 41 shows the influence of murine ANP32B protein point mutant onH7N9_(AH13) polymerase activity.

SPECIFIC MODE FOR CARRYING OUT THE INVENTION

The present invention is described in detail below with reference to theexamples and the accompanying drawings. It will be understood by thoseskilled in the art that the following examples are for illustrativepurposes and should not be construed as limiting the present inventionin any way. The protection scope of the present invention is defined bythe appended claims.

Example 1. Construction of ANP32 Protein Expression Vector

The nucleotide sequences of ANP32 proteins from chicken, human, zebrafinch, duck, turkey, pig, mouse, horse, etc. are as follows:

chicken ANP32A (chANP32A) (Gallus gallus, XM_413932.5), human ANP32A(huANP32A) (Homo sapiens, NM 006305.3), zebra finch ANP32A (zfANP32A)(Taeniopygia guttata, XM_012568610.1), duck ANP32A (dkANP32A) (Anasplatyrhynchos, XM_005022967.1), turkey ANP32A (tyANP32A) (Meleagrisgallopavo, XM_010717616.1), pig ANP32A (pgANP32A) (Sus scrofa,XM_003121759.6), murine ANP32A (muANP32A) (Mus musculus, NM 009672.3),equine ANP32A (eqANP32A) (Equus caballus, XM_001495810.5), chickenANP32B (chANP32B) (Gallus gallus, NM_001030934.1), human ANP32B(huANP32B) (Homo sapiens, NM_006401.2).

The amino acid sequences of ANP32 proteins from chicken, human, zebrafinch, duck, turkey, pig, mouse, horse, etc. are as follows:

chicken ANP32A (chANP32A) (Gallus gallus, XP_413932.3), human ANP32A(huANP32A) (Homo sapiens, NP_006296.1), zebra finch ANP32A (zfANP32A)(Taeniopygia guttata, XP_012424064.1), duck ANP32A (dkANP32A) (Anasplatyrhynchos, XP_005023024.1), turkey ANP32A (tyANP32A) (Meleagrisgallopavo, XP_010715918.1), pig ANP32A (pgANP32A) (Sus scrofa,XP_003121807.3), mouse ANP32A (muANP32A) (Mus musculus, NP_033802.2),equine ANP32A (eqANP32A) (Equus caballus, XP_001495860.2), chickenANP32B (chANP32B) (Gallus gallus, NP_001026105.1), human ANP32B(huANP32B) (Homo sapiens, NP_006392.1).

First, a PCAGGS-Flag recombinant plasmid was constructed. A start codon(ATG) was introduced at the N-terminus of the Flag-tag(GGCAGCGGAGACTACAAGGATGACGATGACAAG, SEQ ID NO:1), and a stop codon (TGA)was introduced at the C-terminus; NotI (GCGGCCGC, SEQ ID NO:2)restriction site was introduced upstream of the start codon, and XhoI(CTCGAG, SEQ ID NO:3) restriction site was introduced downstream of thestop codon, and a 15 bp homologous arm (underlined) of PCAGGS vector wasintroduced at the outer ends of the two restriction sites, and twoprimers Flag-S(SEQ ID NO: 4) and Flag-A (SEQ ID NO: 5) complementary tothe Flag-tag gene fragment were synthesized.

Flag-S: SEQ ID NO: 4 5-AAAGAATTCGAGCTC GCGGCCGCATGGGCAGCGGAGACTACAAGGATGACGATGACAAGTGACTCGAG CTAGCAGATCTTTTT-3 Flag-A: SEQ ID NO: 55-AAAAAGATCTGCTAG CTCGAGTCACTTGTCATCGTCATCCTTGTAGT CTCCGCTGCCCATGCGGCCGCGAGCTCGAATTCTTT-3

The designed upstream and downstream primers Flag-S and Flag-A werediluted respectively to 100 uM (diluted with TE buffer); 10 ul of eachdiluted primers were taken and then mixed uniformly, and placed at 95°C. for 5 min; the PCR instrument was turned off, and the temperature wasnaturally reduced to room temperature (about 2 h), and the obtainedproduct is an annealed synthetic sample. Commercially available PCAGGSvector (see FIG. 1, purchased from Fenghui Bio, product number V00514,cat # JM004, www.fenghbio.cn) was subjected to double enzyme digestionby Thermo rapid restriction endonuclease Not I and Xho I under a waterbath condition at 37° C. for 1.5 h, and the enzyme digested fragmentswere recovered by using a gel recovery kit (OMEGA, cat # D2500-01), andthe obtained product is recovered for future use. The PCAGGSdouble-enzyme digested product and the annealed synthetic sample wereligated by In-Fusion ligase (purchased from Clontech, cat #639648)according to the instructions, and then transformed into DH5a competentcells. The next day, a single clone was selected and sequenced, and theplasmid which was verified correct by sequencing was named asPCAGGS-Flag plasmid which was used as a control plasmid for subsequentexperiments and was extracted in large-scale for later use.

According to the nucleotide sequences of ANP32A and ANP32B of eachspecies mentioned above, the sequences of ANP32A and ANP32B of eachspecies mentioned above were synthesized respectively. During thesynthesis, the stop codon was removed at the C-terminus of the genefragment and the a Flag-tag (SEQ ID NO: 1) was added in tandem; a stopcodon (TGA) was added at the end of the Flag-tag; Not I (SEQ ID NO: 2)and Xho I (SEQ ID NO: 3) restriction sites were introduced at both endsof the synthesized fragment. The PCAGGS-Flag vector was digested byThermo rapid restriction endonucleases Not I and Xho I at 37° C. for 1.5h, and the digested fragments were recovered using a gel recovery kit(OMEGA, cat # D2500-01). The PCAGGS-Flag double-digested product andeach gene fragment were ligated by In-Fusion ligase (purchased fromClontech, cat #639648) according to the instructions, and thentransformed into DH5a competent cells. The next day, a single clone wasselected and sequenced, and the fragment was finally inserted into thePCAGGS vector. For example, the plasmid map of PCAGGS-huANP32A is shownin FIG. 2. The plasmid maps of ANP32A and ANP32B genes of other speciesare similar to that of FIG. 2, and only the corresponding gene sequencesare replaced. For example, the plasmid map of PCAGGS-huANP32B is asequence in which huANP32A is replaced by huANP32B.

After the recombinant plasmids were sequenced correctly, 1 ug of therecombinant plasmids were respectively transfected into 293T cells bylipofectamine 2000 reagent, and the cell lysate was taken after 48hours; the protein expression was detected by Flag-antibody (purchasedfrom Sigma, cat # F1804-1MG) by utilizing western blotting;intracellular β-actin was used as an internal control gene; the antibodyMonoclonal Anti-β-Actin antibody produced in mouse (purchased fromSigma, cat # A1978-200UL) was used and the result was shown in FIG. 3showing that ANP32A and ANP32B proteins from various species were wellexpressed.

Example 2: Construction of a Cell Line

We performed the construction of cell line by using CRISPR-Cas9technology. According to NCBI published reference nucleotide sequencesof human ANP32A (NM_006305.3) and human ANP32B (NM_006401.2), sgRNAs forthe two proteins were designed by using the online softwarehttp://crispr.mit.edu/ (see Table 1 for sequences).

TABLE 1 sgRNA sequences primer name primer sequence (5′-3′)human ANP32A-sgRNA, TCTTAAGTACAATCAACGT SEQ ID NO: 6 human ANP32B-sgRNA,GCCTACATTTATTAAACTG SEQ ID NO: 7

The pMD18T-U6 recombinant plasmid, which contains a human U6 promotersequence+sgRNA sequence (huANP32A or huANP32B)+sgRNA scaffoldsequence+TTTTTT, was constructed as follows.

First, a gene fragment is synthesized, wherein the fragment contains ahuman U6 promoter sequence+huANP32A-sgRNA sequence+sgRNA scaffoldsequence+TTTTTT, and the sequence is SEQ ID NO: 8. The synthesizedfragment was directly ligated into a pMD-18T vector (TaKaRa, Cat. No.D101A), and a pMD18T-U6-huANPsgRNA-1 recombinant plasmid (containinghuANP32AsgRNA) was successfully constructed. Using this plasmid as atemplate, sgRNA primers for huANP32B were designed, and amplified byKOD-FX Neo high-efficiency DNA polymerase (Cat. No.: KFX-201, purchasedfrom Toyobo) using the overlapping PCR method (the reaction conditionand reaction system were based on the instructions of said polymerase;unless otherwise specified, except for the quantitative PCR reaction,KOD-FX Neo high-efficiency DNA polymerase was used in the PCR reactionsof the following examples, and the reaction system and reactioncondition were based on the instructions of said polymerase), toconstruct a plasmid containing sgRNA of huANP32B; and the primersequences were shown in Table 2, and the PCR system and procedure wereperformed with reference to the KOD-FX Neo instructions. The obtainedPCR product was digested with Dpn I in a 37° C. constant temperaturewater bath for 30 minutes, and then 5 ul of the digested product wastaken to be transformed into 20 ul of DH5α competent cells; the nextday, a single clone was picked for sequencing, and the plasmid which wasverified correct by sequencing, namely pMD18T-U6-huANPsgRNA-2(containing huANP32BsgRNA), was used for subsequent transfectionexperiment.

TABLE 2 primer sequences primer name primer sequence (5′-3′)huANP32B-sgRNA-F, CGGGTCCGGTTCCTCAGCTCCGGTGTTTCGT SEQ ID NO: 9 CChuANP32B-sgRNA-R, GGAGCTGAGGAACCGGACCCGTTTTAGAGCT SEQ ID NO: 10 AG

1 ug of eukaryotic plasmid pMJ920 (Addge plasmid #42234) expressingCas9-GFP protein and pMD18T-U6-huANPsgRNA-1 or pMD18T-U6-huANPsgRNA-2recombinant plasmids were taken respectively, and mixed withlipofectamine 2000 at a ratio of 1:2.5, and then transfected into 293Tcells. After 48 hours, GFP-positive cells were screened by anultra-speed flow cytometry sorting system, and plated in a 96-well plateat a single cell/well for about 10 days; single-cell clones were pickedfor expansion and culture, and then cellular RNA was extracted accordingto the procedure using a SimplyP total RNA extraction Kit (purchasedfrom Bioflux, cat # BSC52M1), and cDNA was synthesized using a reversetranscription Kit of Takara Co., Ltd (PrimneScript™ RT reagent Kit withgDNA Eraser (Perfect read Time), Cat.RR047A); and sgRNA-targetingfragments of huANP32A and huANP32B were amplified by KOD Fx Neopolymerase using the cDNA as the template, and the amplification primerswere shown in Table 3, wherein the size of huANP32A amplified fragmentwas 390 bp, and the size of huANP32B amplified fragment was 362 bp.Single-cell clones that were verified as gene deletion by sequencingwere subject to western blotting and fluorescent quantitativeidentification. The huANP32A and huANP32B double-knockout cell lineswere obtained after the first round of obtaining the huANP32Bsingle-knockout cell line, followed by another round of knockoutscreening, and the transfection system and screening steps were asdescribed above.

TABLE 3 the primer sequences for identification ofhuANP32A and huANP32B knockout cell line primer sequence primer name(5′-3′) QhuANP32A-F180, GGGCAGACGGATTCATTTAGAG SEQ ID NO: 11QhuANP32A-R570, TTCTCGGTAGTCGTTCAGGTTG SEQ ID NO: 12 QhuANP32B-F312,GCGGAAAGTTAAGTTTGAAGAG SEQ ID NO: 13 G QhuANP32B-R674,GCGGAAAGTTAAGTTTGAAGAG SEQ ID NO: 14 G

Anti-PHAP1 antibody (purchased from Abcam, cat # ab51013) andAnti-PHAPI2/APRIL antibody [EPR14588] (purchased from Abcam, cat #ab200836) were used in Western bloting; β-actin was used as the internalcontrol gene, and the antibody of Monoclonal Anti-β-Actin antibodyproduced in mouse (purchased from Sigma, cat # A1978-200UL) was used,and the results were shown in FIG. 4. The primers for the fluorescentquantitative identification of the knockout cell line were shown inTable 4; β-actin was used as an internal control gene; the results ofthe fluorescent PCR identification were shown in FIG. 5. Based on theabove, we successfully constructed a huANP32A single-knockout cell line(AKO), a huANP32B single-knockout cell line (BKO), and a huANP32A andANP32B double-knockout cell line (DKO), which were used for subsequentexperiments. Fluorescent quantitative PCR was performed usingSYBR®Premix Ex Taq™ II (Tli RnaseH plus) (Cat. # RR820A) produced byTAKARA according to the instructions (the subsequent fluorescentquantitative PCR was also performed using the kit produced by TAKARA).

TABLE 4 the primer sequences for fluorescencequantitation of huANP32A and huANP32B primer sequence primer name(5′-3′) qhu32A-F1, GGCAGACGGATTCATTTAGAGC SEQ ID NO: 15 qhu32A-R,CTTTGGTAAGTTTGCGATTGA SEQ ID NO: 16 qhu32B-F, CTGCCCCAGCTTACCTACTTGSEQ ID NO: 17 qhu32B-R, ATCCTCATCGTCCTCGTCTTC SEQ ID NO: 18 actin-F,CATCTGCTGGAAGGTGGACAA SEQ ID NO: 19 actin-R, CGACATCCGTAAGGACCTGTASEQ ID NO: 20

Example 3: Detection of Influenza Polymerase Activity

The influenza polymerase reporter system involved in the presentinvention includes influenza polymerases PB2, PB1 and PA proteins, and anuclear protein NP. These proteins are derived from human influenza H1N1subtype A/Sichuan/01/2009 (H1N1_(SC09)) and A/WSN/1933(WSN), humaninfluenza H7N9 subtype A/Anhui/01/2013 (H7N9_(AH13)), and canineinfluenza H3N2 subtype A/canine/Guangdong/1/2011 (H3N2_(GD11)), avianinfluenza H9N2 subtype A/chicken/Zhejiang/B2013/2012 (H9N2_(ZJ12)) andH7N9 subtype A/chicken/Zhejiang/DTID-ZJU01/2013(H7N9_(ZJ13)), equineinfluenza A/equine/Jilin/1/1989 (H3N8_(JL89)) andA/equine/Xinjiang/3/2007 (H3N8_(XJ07)). The sequences of PB2, PB1, PAand NP proteins of these influenza subtypes are shown in Table 5.

TABLE 5 The nucleotide sequences of PB2, PB1, PA and NP proteins NP ofhuman H1N1_(sc09) (Genebank: GQ166225.1) PA of human H1N1_(sc09)(Genebank: GQ166226.1) PB1 of human H1N1_(sc09) (Genebank: GQ166227.1)PB2 of human H1N1_(sc09) (Genebank: GQ166228.1) PB2 of human H1N1_(WSN)(Genebank: CY034139.1) PB1 of human H1N1_(WSN) (Genebank: CY034138.1) PAof human H1N1_(WSN) (Genebank: CY034137.1) NP of human H1N1_(WSN)(Genebank: CY034135.1) PB2 of human H7N9_(AH13) (Genebank: EPI439504)PB1 of human H7N9_(AH13) (Genebank: EPI439508) PA of human H7N9_(AH13)(Genebank: EPI439503) NP of human H7N9_(AH13) (Genebank: EPI439505) PB2of canine H3N2_(GD11) (Genebank: JX195347.1) PB1 of canine H3N2_(GD11)(Genebank: JX195346.1) PA of canine H3N2_(GD11) (Genebank: JX195340.1)NP of canine H3N2_(GD11) (Genebank: JX195341.1) PB2 of avian H9N2_(ZJ12)(Genebank: KP865886.1) PB1 of avian H9N2_(ZJ12) (Genebank: KP865839.1)PA of avian H9N2_(ZJ12) (Genebank: KP865793.1) NP of avian H9N2_(ZJ12) (Genebank: KP865771.1) PB2 of equine H3N8_(JL89) (Genebank: KF285454.1)PB1 of equine H3N8_(JL89) (Genebank: KF285455.1) PA of equineH3N8_(JL89) (Genebank: KF285456.1) NP of equine H3N8_(JL89) (Genebank:M63786.1) PB2 of equine H3N8_(XJ07) (Genebank: EU794556.1) PB1 of equineH3N8_(XJ07) (Genebank: EU794557.1) PA of equine H3N8_(XJ07) ( Genebank:EU794558.1) NP of equine H3N8_(XJ07) (Genebank: EU794560.1) PB2 of avianH7N9_(ZJ13) (Genebank: KC899666.1) PB1 of avian H7N9_(ZJ13) (Genebank:KC899667.1) PA of avian H7N9_(ZJ13) (Genebank: KC899668.1) NP of avianH7N9_(ZJ13) (Genebank: KC899670.1)

A plasmid containing the above-mentioned proteins of each influenzavirus subtype was constructed, for example, H1N1_(SC09) polymerasecontained PB2, PB1 and PA proteins and a nuclear protein NP derived fromhuman influenza H1N1 subtype A/Sichuan/01/2009 (H1N1_(SC09)), and wasnamed as the H1N1_(SC09) polymerase reporter system; plasmids wereconstructed with the vector PCAGGS for PB2, PB1, PA and NP,respectively, and were named as PB2 plasmid, PB1 plasmid, PA plasmid,and NP plasmid. The same is for others.

Taking the H1N1_(SC09) polymerase reporter system as an example, theconstruction process was as follows:

mRNA of H1N1_(SC09) strain (Master's thesis of Zhang Qianyi,“Establishment of reverse genetic operating system for H1N1 influenzavirus A/Sichuan/01/2009 strain”, Gansu Agricultural University, 2011)was extracted according to the operation manual of QIAamp Viral RNA MiniKit (purchased from QIAGEN, cat #52904), and then cDNA was synthesizedaccording to the instruction of M-MLV reverse transcriptase kit(purchased from Invitrogen, cat #28025-013) using Uni12 (AGCAAAAGCAGG,SEQ ID NO:21) as reverse transcription primer. Based on the sequenceinformation of each gene fragment, PCR primers were designed (see Table7); a 15 bp PCAGGS homologous arm was respectively introduced at bothends; the gene of interest was synthesized using KOD FX Neohigh-efficiency polymerase; and then the amplified fragment of each geneof H1N1_(SC09) and the double-digested PCAGGS vector in Example 1 wereligated at room temperature for 30 minutes using the seamless cloningkit, ClonExpress II One Step Cloning Kit (purchased from Vazyme, cat #C112-01) according to the instructions; the ligation product wastransformed into 20 ul DH5α competent cells, and the next day a singleclone was picked for sequencing. The plasmid which were verified correctby sequencing were respectively named as PB2 plasmid, PB1 plasmid, PAplasmid and NP plasmid, and used for subsequent experiments. The same isfor others. See Table 6 for the sources of other various strains.

TABLE 6 Human H1N1 Neumann G; Watanabe T; Ito H; Watanabe S; Goto H; WSNGao P; Hughes M; Perez D R; Donis R; Hoffmann E; Hobom G; Kawaoka Y.Generation of influenza A viruses entirely from cloned cDNAs. [J].Proceedings of the National Academy of Sciences of the United States ofAmerica, 1999, 96(16): 9345-50 human Zhang, Q., Shi, J., Deng, G., Guo,J., Zeng, X., A/Anhui/01/2013 He, X., Kong, H., Gu, C., Li, X., Liu, J.,et al. (H7N9_(AH13)) (2013). H7N9 influenza viruses are transmissible inferrets by respiratory droplet. Science. 341(6144), 410-414 canineinfluenza Su S, Li H T, Zhao F R, et al. Avian-origin H3N2 H3N2 subtypecanine influenza virus circulating in farmed dogs in A/canine/Guangdong, China[J]. Infection Genetics & Evolution, Guangdong/ 2013,14(2): 444-449 1/2011 (H3N2_(GD11)) avian influenza Teng Q, Xu D, ShenW, et al. A Single Mutation at H9N2 subtype Position 190 inHemagglutinin Enhances Binding A/chicken/ Affinity for Human Type SialicAcid Receptor and Zhejiang/ Replication of H9N2 Avian Influenza Virus inB2013/2012 Mice[J], Journal of Virology, 2016, 90(21): (H9N2_(ZJ12))9806 avian Li C, Li C, Zhang A J, et al. Avian influenza A H7N9 H7N9subtype virus induces severe pneumonia in mice without prior A/chicken/adaptation and responds to a combination of zanamivir Zhejiang/ andCOX-2 inhibitor[J]. Plos One, 2014, 9(9): DTID-ZJU01/ e1079662013(H7N9_(ZJ13)) equine Zhang Xiang, Guo Wei, Wang Xiaojun.Construction A/equine/ of Two-way Transcription/Expression Vector andJilin/1/1989 Its Application in Reverse Genetic System of (H3N8_(JL89))Equine Influenza Virus [J]. Chinese Journal of Preventive VeterinaryMedicine, 2016, 38 (11): 860-864 equine master's thesis A/equine/ DaiLingli. Sequence analysis of HA gene of equine Xinjiang/3/ influenzavirus A/Equine/Xinjiang/3/07 (H3N8) and 2007 establishment of two PCRdetection methods [D]. (H3N8_(XJ07)) Chinese Academy of AgriculturalSciences, 200

TABLE 7 Polymerase construction primers: primer name primer sequenceH1N1 TTCGAGCTCGCGGCCGCATGGAGAGAATAAAAG SC09-PB2-F AACT SEQ ID NO: 22H1N1 ATCTGCTAGCTCGAGCTAATTGATGGCCATCCGA SC09-PB2-R A SEQ ID NO: 23 H1N1TTCGAGCTCGCGGCCGCATGGATGTCAATCCGAC SC09-PB1-F TCT SEQ ID NO: 24 H1N1ATCTGCTAGCTCGAGTTATTTTTGCCGTCTGAGT SC09-PB1-R T SEQ ID NO: 25 H1N1TTCGAGCTCGCGGCCGCATGGAAGACTTTGTGCG SC09-PA-F AC SEQ ID NO: 26 H1N1ATCTGCTAGCTCGAGCTACTTCAGTGCATGTGTG SC09-PA-R A SEQ ID NO: 27 H1N1TTCGAGCTCGCGGCCGCATGGCGTCTCAAGGCA SC09-NP-F CCAA SEQ ID NO: 28 H1N1ATCTGCTAGCTCGAGTCAACTGTCATACTCCTCT SC09-NP-R G SEQ ID NO: 29 H1N1TTCGAGCTCGCGGCCGCATGGAAAGAATAAAAG WSN-PB2-F AAC SEQ ID NO: 30 H1N1ATCTGCTAGCTCGAGCTATTCGACACTAATTGAT WSN-PB2-R G SEQ ID NO: 31 H1N1TTCGAGCTCGCGGCCGCATTTGAATGGATGTCAA WSN-PB1-F TCCGAC SEQ ID NO: 32 H1N1ATCTGCTAGCTCGAGTCATGAAGGACAAGCTAA WSN-PB1-R ATTCA SEQ ID NO: 33 H1N1TTCGAGCTCGCGGCCGCCTGATTCAAAATGGAA WSN-PA-F GATT SEQ ID NO: 34 H1N1ATCTGCTAGCTCGAGTTTTTGGACAGTATGGATA WSN-PA-R GCAAA SEQ ID NO: 35 H1N1TTCGAGCTCGCGGCCGCTCACTCACAGAGTGAC WSN-NP-F ATCGA SEQ ID NO: 36 H1N1ATCTGCTAGCTCGAGTTCTTTAATTGTCGTACTC WSN-NP-R CT SEQ ID NO: 37 H7N9TTCGAGCTCGCGGCCGCATGGAAAGAATAAAAG AH13-PB2-F AAC SEQ ID NO: 38 H7N9ATCTGCTAGCTCGAGTTAATTGATGGCCATCCGA AH13-PB2-R AT SEQ ID NO: 39 H7N9TTCGAGCTCGCGGCCGCATGGATGTCAATCCGAC AH13-PB1-F TTT SEQ ID NO: 40 H7N9ATCTGCTAGCTCGAGCTATTTTTGCCGTCTGAGC AH13-PB 1-R TC SEQ ID NO: 41 H7N9TTCGAGCTCGCGGCCGCATGGAAGACTTTGTGCG AH13-PA-F AC SEQ ID NO: 42 H7N9ATCTGCTAGCTCGAGCTATCTTAGTGCATGTGTG AH13-PA-R A SEQ ID NO: 43 H7N9TTCGAGCTCGCGGCCGCATGGCGTCTCAAGGCA AH13-NP-F CCA SEQ ID NO: 44 H7N9ATCTGCTAGCTCGAGTCAATTGTCATACTCCTCT AH13-NP-R GC SEQ ID NO: 45 H3N2TTCGAGCTCGCGGCCGCATGGAGAGAATAAAAG GD12-PB2-F AATT SEQ ID NO: 46 H3N2ATCTGCTAGCTCGAGCTAATTGATGGCCATCCGA GD12-PB2-R A SEQ ID NO: 47 H3N2TTCGAGCTCGCGGCCGCATGGATGTCAATCCGAC GD12-PB1-F TTT SEQ ID NO: 48 H3N2ATCTGCTAGCTCGAGCTATTTTTGCCGTCTGAGC GD12-PB1-R TC SEQ ID NO: 49 H3N2TTCGAGCTCGCGGCCGCATGGAAGACTTTGTGCG GD12-PA-F ACAA SEQ ID NO: 50 H3N2ATCTGCTAGCTCGAGCTATTTCAGTGCATGTGTG GD12-PA-R AGG SEQ ID NO: 51 H3N2TTCGAGCTCGCGGCCGCATGGCGTCTCAAGGCA GD12-NP-F CCAAAC SEQ ID NO: 52 H3N2ATCTGCTAGCTCGAGTTAATTGTCATACTCCTCT GD12-NP-R GC SEQ ID NO: 53 H3N8TTCGAGCTCGCGGCCGCATGGAGAGAATAAAAG JL89-PB2-F AATT SEQ ID NO: 54 H3N8ATCTGCTAGCTCGAGCTAATTGATGGCCATCCGA JL89-PB2-R AT SEQ ID NO: 55 H3N8TTCGAGCTCGCGGCCGCATGGATGTCAATCCGAC JL89-PB1-F TTT SEQ ID NO: 56 H3N8ATCTGCTAGCTCGAGTCACTGTTTTTGCCGTCTG JL89-PB1-R AG SEQ ID NO: 57 H3N8TTCGAGCTCGCGGCCGCATGGAAGATTTTGTGCG JL89-PA-F ACAA SEQ ID NO: 58 H3N8ATCTGCTAGCTCGAGCTATTTCAGTGCATGTGTG JL89-PA-R A SEQ ID NO: 59 H3N8TTCGAGCTCGCGGCCGCAGCAAAAGCAGGGTAG JL89-NP-F ATAAT SEQ ID NO: 60 H3N8ATCTGCTAGCTCGAGAGTAGAAACAAGGGTATT JL89-NP-R TTTC SEQ ID NO: 61 H3N8TTCGAGCTCGCGGCCGCATGGAGAGAATAAAAG XJ07-PB2-F AACT SEQ ID NO: 62 H3N8ATCTGCTAGCTCGAGTTAATTGATGGCCATCCGA XJ07-PB2-R AT SEQ ID NO: 63 H3N8TTCGAGCTCGCGGCCGCATGGATGTCAATCCGAC XJ07-PB1-F TCT SEQ ID NO: 64 H3N8ATCTGCTAGCTCGAGCTATTTTTGCCGTCTGAGC XJ07-PB1-R SEQ ID NO: 65 H3N8TTCGAGCTCGCGGCCGCATGGAAGACTTTGTGCG XJ07-PA-F ACA SEQ ID NO: 66 H3N8ATCTGCTAGCTCGAGTTACTTCAGTGCATGTGTA XJ07-PA-R AGG SEQ ID NO: 67 H3N8TTCGAGCTCGCGGCCGCATGGCGTCTCAAGGCA XJ07-NP-F CCAAA SEQ ID NO: 68 H3N8ATCTGCTAGCTCGAGTTAACTGTCAAATTCCTCA XJ07-NP-R GC SEQ ID NO: 69 H9N2TTCGAGCTCGCGGCCGCATGGCGTCTCAAGGCA ZJ12-NP-F C SEQ ID NO: 70 H9N2ATCTGCTAGCTCGAGTCAATTGTCATACTCCT Z112-NP-R SEQ ID NO: 71 H9N2TTCGAGCTCGCGGCCGCATGGAAGACTTTGTGCG Z112-PA-F SEQ ID NO: 72 H9N2ATCTGCTAGCTCGAGCTATCTTAGTGCATGTG Z112-PA-R SEQ ID NO: 73 H9N2TTCGAGCTCGCGGCCGCATGGATGTCAATCCGA Z112-PB1-F SEQ ID NO: 74 H9N2ATCTGCTAGCTCGAGCTATTTTTGCCGTCTGAG Z112-PB1-R SEQ ID NO: 75 H9N2TTCGAGCTCGCGGCCGCATGGAAAGAATAAAAG ZI12-PB2-F A SEQ ID NO: 76 H9N2ATCTGCTAGCTCGAGTTAATTGATGACCATCCG Z112-PB2-R SEQ ID NO: 77

We constructed a reporter plasmid (pMD18T-vLuc) for detecting theactivity of polymerase; the reporter plasmid uses the pMD18T plasmid asthe backbone and contains the sequence of interest; the sequence ofinterest is characterized in that the 5′ end non-coding region(sequence: agcaaaagcagggg, SEQ ID NO:78) and the 3′ end non-codingregion (sequence: gtatactaataattaaaaacacccttgtttctact, SEQ ID NO:79) ofHA gene of H3N8_(JL89) strain were introduced into both ends of theprotein coding sequence of Firefly luciferase; a human polI promoter wasintroduced into the 3 ‘end of the sequence, and a murine pol Iterminator sequence was introduced into the 5′end of the sequence. Thesequence of interest is as follows: murine pol I terminator sequence(bold underlined)+5’ non-coding region (red italics) of HA gene+genesequence of Firefly luciferase+3′ non-coding region (green italics) ofHA gene+human polI promoter (bold underlined), SEQ ID NO: 80. Thesynthesized fragment was directly ligated into the pMD18-T vector,obtaining the reporter plasmid pMD18T-vLuc.

After co-transfecting this reporter plasmid and the influenza polymerasesystem into 293T, the polymerase complex can recognize the non-codingsequences of the virus at both ends of Firefly luciferase, therebystarting the synthesis of Firefly luciferase gene vRNA, cRNA and mRNA.In order to make the polymerase system more stable and stringent, weintroduced a polymerase dual fluorescence reporter system, whereinRenilla luciferase (pRL-TK) was further added as an internal controlinto the above influenza polymerase reporter system, and we establisheda stable transfection system: taking a 12-well plate as an example,adding PB1 plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NPplasmid (160 ng), pMD18T-vLuc plasmid (80 ng) and pRL-TK plasmid (10 ng,Promega cat # E2241, GenBank number: AF025846) into each well, and thentransfecting with the transfection reagent lipo2000. After 24 hours oftransfection, the cell supernatant was discarded; the cells were lysedby 100 ul of cell lysis buffer/well (passive lysis buffer, derived fromthe dual luciferase kit (Promega)), and then measured by the dualluciferase kit (Promega) using Centro. XS LB 960 luminometer (Bertholdtechnologies). Renilla luciferase was used as an internal control, andthe ratio can represent the activity of the polymerase.

We transfected the H1N1_(SC09) polymerase into the cell lines AKO, BKO,DKO constructed in Example 2 and wild-type 293T cell by the abovesystem; each group was set up with triplicate wells and then detected 24hours after transfection, the activity of H1N1_(SC09) polymerase in AKOand BKO was not different from that of wild-type 293T cells, while theactivity of H1N1_(SC09) polymerase in DKO cells decreased by more than10,000 times, see FIG. 6. The results were processed by the biologicalsoftware GraphPad Prism 5 (https://www.graphpad.com) and analyzed byone-way ANOVA and Dunnett's t-test; the difference between eachexperimental group and the control group is shown in the chart: ns meansno difference, * means P<0.05, ** means P<0.01, *** means P<0.001, ****means P<0.0001. The symbols ns, *, **, *** and * * * * in other figuresrelating to the detection of polymerase activity have the same meaningsas above, and processed as above.

H7N9_(AH13), WSN, H3N2_(GD11), H3N8_(JL89), H3N8_(XJ07) polymerase weretransfected into different cell lines AKO, BKO, DKO and wild-type 293Tcells in the same way as H1N1_(SC09) polymerase; the result is that theactivity in AKO and BKO was not different from that in wild-type 293Tcells, while the activity in DKO cells decreased by about 10,000 times;the results are shown in FIG. 7A-E.

The huANP32A and huANP32B expression plasmids constructed in Example 1PCAGGS-huANP32A and PCAGGS-huANP32B were co-transfected with H1N1_(SC09)polymerase into DKO cells; the specific transfection system was: takinga 12-well plate as an example, each well was added with H1N1_(SC09) PB1plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid(160 ng), pMD18T-vLuc plasmid (80 ng), pRL-TK plasmid (10 ng) anddifferent doses of ANP32A and ANP32B proteins, and the specific doses ofANP32A and ANP32B protein plasmids were respectively selected from thefollowing doses: 0 pg, 10 pg, 100 pg, 1 ng, 5 ng, 10 ng, 20 ng, 100 ng,500 ng, 1 ug, and a PCAGGS-Flag empty vector control was set at the sametime; the total amount of plasmid was made up by the PCAGGS-Flag emptyvector, and each group was provided with triplicate wells. Cells werelysed 24 h after transfection, and the polymerase activity was detectedas described above.

The results showed that the supplementation of huANP32A and ANP32Bproteins can restore the activity of polymerase in a dose-dependentmanner, reaching a plateau phase at 20 ng, and the activity of thepolymerase can be restored by about 3000 times. The activity curves ofhuANP32A and huANP32B proteins have the same trend, and there was noadditive effect during the plateau phase, as shown in FIG. 8. Therefore,the polymerase was dose-dependent on huANP32A and ANP32B proteins, andthe dose requirement was low. Whereas, the activity of polymerase wasinhibited when the amount of the huANP32A and huANP32B proteins wasexcessive, as shown in FIG. 9A. DKO cells were co-transfected with thehuANP32A and huANP32B expression plasmids constructed in Example 1 atdifferent doses described above as well as H7N9_(AH13) polymerase, andthe result was similar to H1N1_(SC09), as shown in FIG. 9B.

Example 4: Fluorescent Quantitative Detection of the Influence of ANP32Protein on RNA Synthesis of Influenza Virus

Real-time PCR was used to detect the differences in the synthesis ofcRNA, vRNA and mRNA of influenza virus on wild-type 293T and thedouble-knockout cell line DKO. First, wild-type 293T and double-knockoutcell line DKO cell were plated in a 12-well plate, and the 293T cellswere transfected with the H1N1_(SC09) polymerase dual fluorescencereporter system of Example 3. The transfection system was: PB1 plasmid(80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid (160 ng),pMD18T-vLuc plasmid (80 ng), pRL-TK plasmid (10 ng), and empty vectorPCAGGS-Flag plasmid (20 ng); the DKO cells were transfected withH1N1_(SC09) polymerase dual fluorescence reporter system of Example 3.The transfection system was: PB1 plasmid (80 ng), PB2 plasmid (80 ng),PA plasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80 ng),pRL-TK plasmid (10 ng), and PCAGGS-huANP32A plasmid (20 ng), while theempty vector PCAGGS-Flag plasmid (20 ng) was set as a negative control.After 24 h, total cellular RNA was extracted, and reverse transcriptionwas performed using particular primers (see Table 8) to synthesize thefirst cDNAs of cRNA, vRNA and mRNA, respectively, followed byfluorescent quantitative PCR using specific primers (see Table 9); usingrandom primers (contained in the kit) as reverse transcription primersfor internal control gene β-actin, and the fluorescent quantitativeprimers of β-actin were shown in Table 4 of Example 2. The resultsshowed that compared to the wild type, the synthesis of viral cRNA, vRNAand mRNA was significantly reduced (about 30-50 times) on thedouble-knockout cell line, indicating that RNA of influenza virus washardly replicated in cell lines lacking ANP32A and ANP32B. When humanANP32A was supplemented to the double-knockout cell line, thereplication and synthesis of RNA could be restored, as shown in FIG. 10.The above experimental procedure was repeated with the huANP32B plasmidinstead of the huANP32A plasmid; it was found that human ANP32B also hadthe same function (data is similar to DKO+huANP32A in FIG. 10). Thisshowed that the ANP32A and ANP32B proteins were involved in thesynthesis and replication of influenza virus RNA and played a decisiverole therein.

TABLE 8 Reverse transcription primers primer name primer sequenceLuc-vRNA, CATTTCGCAGCCTACCGTGGTGT SEQ ID NO: 81 T Luc-cRNA,AGTAGAAACAAGGGTG SEQ ID NO: 82 Luc-mRNA, oligo-dT20 SEQ ID NO: 83

TABLE 9 fluorescent quantitative PCR primers primer name primer sequenceLuc-F, SEQ ID NO: 84 GATTACCAGGGATTTCAGTCG Luc-R, SEQ ID NO: 85GACACCTTTAGGCAGACCAG

Example 5: Influence of ANP32 Protein on the Replication of InfluenzaVirus

Double-knockout cell lines (DKO) were plated in a 12-well plate at3×10⁵/well; after 20 hours, the ANP32A and ANP32B protein plasmids ofdifferent species constructed in Example 1 were co-transfected with 6plasmids of the H1N1_(SC09) polymerase reporter system. The transfectionsystem was: PB1 plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40ng), NP plasmid (160 ng), pMD 18T-vLuc plasmid (80 ng), pRL-TK plasmid(10 ng), and ANP32A or ANP32B protein plasmid (20 ng), and the emptyvector PCAGGS-Flag (20 ng) was set as a negative control, and each groupwas provided with triplicate wells. 24 h after transfection, the cellswere lysed as described in Example 3 to detect the activity ofpolymerase, showing that: compared to the empty vector, avian ANP32Asuch as chANP32A, dkANP32A, zfANP32A, tyANP32A, mammalian ANP32 such ashuANP32A, pgANP32A, eqANP32A, huANP32B all supported the activity ofH1N1_(SC09) polymerase, whereas the two proteins chANP32B and muANP32Adid not have the ability to support the activity of H1N1_(SC09)polymerase, as shown in FIG. 11A.

The above experiment was repeated with the H7N9_(AH13) polymerasereporter system instead of the H1N1_(SC09) polymerase reporter system,and the result showed that: compared to the empty vector, avian ANP32Asuch as chANP32A, dkANP32A, zfANP32A, tyANP32A, mammalian ANP32 such ashuANP32A, pgANP32A, eqANP32A, huANP32B all supported the activity ofH7N9_(AH13) polymerase, whereas the two proteins chANP32B and muANP32Adid not have the ability to support the activity of H7N9_(AH13)polymerase, as shown in FIG. 11B.

The above experiment was repeated with the H3N2_(G)m1 polymerasereporter system instead of the H1N1_(SC09) polymerase reporter system,and the result showed that: compared to the empty vector, avian ANP32Asuch as chANP32A, dkANP32A, zfANP32A, tyANP32A, mammalian ANP32 such ashuANP32A, pgANP32A, eqANP32A, huANP32B all supported the activity ofH3N2_(G)m1 polymerase, whereas the two proteins chANP32B and muANP32Adid not have the ability to support the activity of H3N2_(G)m1polymerase, as shown in FIG. 11C.

The above experiment was repeated with the H3N8_(XJ07) polymerasereporter system instead of the H1N1_(SC09) polymerase reporter system,and the result showed that: compared to the empty vector, avian ANP32Asuch as chANP32A, dkANP32A, zfANP32A, tyANP32A, mammalian ANP32 such ashuANP32A, pgANP32A, eqANP32A, huANP32B all supported the activity ofH3N8_(XJ07) polymerase, whereas the two proteins chANP32B and muANP32Adid not have the ability to support the activity of H3N8_(XJ07)polymerase, as shown in FIG. 11D.

The above experiment was repeated with the H3N8_(JL89) polymerasereporter system instead of the H1N1_(SC09) polymerase reporter system,and the result showed that: compared to the empty vector, avian ANP32Asuch as chANP32A, dkANP32A, zfANP32A, tyANP32A supported the activity ofH3N8_(JL89) polymerase, whereas chANP32B and mammalian ANP32 such ashuANP32A, pgANP32A, eqANP32A, huANP32B, muANP32A did not support theactivity of H3N8_(JL89) polymerase, as shown in FIG. 11E.

The above experiment was repeated with the H9N2_(JL12) polymerasereporter system instead of the H1N1_(SC09) polymerase reporter system,and the result showed that: compared to the empty vector, avian ANP32Asuch as chANP32A, dkANP32A, zfANP32A, tyANP32A supported the activity ofH9N2_(JL12) polymerase, whereas chANP32B and mammalian ANP32 such ashuANP32A, eqANP32A, huANP32B, muANP32A did not support the activity ofH9N2_(JL12) polymerase, and pgANP32A substantially did not support theactivity of H9N2_(ZJ12) polymerase. The results were shown in FIG. 11F.

The above experiment was repeated with the WSN polymerase reportersystem instead of the H1N1_(SC09) polymerase reporter system, and theresult showed that: compared to the empty vector, avian ANP32A such aschANP32A, dkANP32A, zfANP32A, tyANP32A, mammalian ANP32 such ashuANP32A, pgANP32A, eqANP32A, huANP32B all supported the activity of WSNpolymerase, whereas the two proteins chANP32B and muANP32A did not havethe ability to support the activity of WSN polymerase, as shown in FIG.11G.

Example 6: H1N1_(SC09) Influenza Virus Transfected Cell Line

The influence of ANP32A and ANP32B proteins on virus replication wasfurther investigated using the influenza virus reverse genetic system.Eight gene fragments of influenza H1N1_(SC09) (PB2, PB1, PA, NP, HA, NA,M and NS) were ligated into a pBD vector with a double promoter (Journalof Virology, September 2005, p12058-12064, Molecular Basis ofReplication of Duck H5N1 influencza Viruses in a Mammalian Mouse Model;“Establishment of reverse genetic operating system for H1N1 influenzavirus A/Sichuan/01/2009 strain”, Zhang Qianyi et al, Veterinary Sciencein China, 2011, 41 (05): 448-452).

The steps for constructing the pBD vector were as follows: the Pol-HDVRexpression cassette was inserted in reverse orientation into the XbaIcleavage site, using pCI vector (purchased from Promega, Cat. No. E1841,GenBank No. U47120) as the backbone. The sequence of the Pol-HDVRexpression cassette is artificially synthesized as SEQ ID NO: 86. Thatis, the pCI vector was digested with XbaI restriction enzyme (NEB, cat #R0145S), and then the linearized vector was recovered using a gelrecovery kit (OMEGA, cat # D2500-01), treated with dephosphorylatingenzyme CIAP (purchased from TAKARA, cat # D2250) according to theinstructions, and then recovered for use. The artificially synthesizedPol-HDVR expression cassette and the digested fragments of pCI vectorhave a 15 bp homologous arm on both the left arm and the right arm, thenthe Pol-HDVR expression cassette fragment and the pCI linearized vectorwere ligated for 30 min at room temperature by using a seamless cloningKit of Clonexpress II One Step Cloning Kit (purchased from Vazyme, cat #C112-01) according to the instructions; the ligation product wastransformed into 20 ul of DH5α competent cells, and the next day asingle clone was picked for sequencing. The plasmid which was verifiedcorrect by sequencing was the pBD two-way expression vector.

According to the methods described in Zhang Qianyi et al, “Establishmentof reverse genetic operating system for H1N1 influenza virusA/Sichuan/01/2009 strain”, Veterinary Science in China, 2011, 41(05):448-452 and Master's thesis of Zhang Qianyi, “Establishment of reversegenetic operating system for H1N1 influenza virus A/Sichuan/01/2009strain”, Gansu Agricultural University, 2011, the H1N1_(SC09) pBD 8plasmid system was constructed as follows:

mRNA of H1N1_(SC09) strain was extracted according to the operationmanual of QIAamp Viral RNA Kit Manual, and then cDNA was synthesizedusing Uni12 (AGCAAAAGCAGG, SEQ ID NO:21) as a reverse transcriptionprimer according to the instructions of Invitrogen M-MLV Kit. Based onthe sequence information of each gene fragment (the sequence of PB2 isSEQ ID NO: 87, the sequence of PB1 is SEQ ID NO: 88, the sequence of PAis SEQ ID NO: 89, the sequence of NP is SEQ ID NO: 90, and the sequenceof HA is SEQ ID NO: 91, the sequence of NA is SEQ ID NO: 92, thesequence of M is SEQ ID NO: 93 and the sequence of NS is SEQ ID NO: 94),PCR primers were designed (see Table 10); a 15 bp pBD homologous arm wasrespectively introduced at both ends; the gene of interest andlinearized pBD vector were amplified using KOD FX Neo high-efficiencypolymerase; and then the amplified fragment of each gene of H1N1_(SC09)and the linearized pBD vector were ligated at room temperature for 30minutes by using the seamless cloning kit of ClonExpress II One StepCloning Kit (purchased from Vazyme, cat # C112-01) according to theinstructions; the ligation product was transformed into 20 ul DH5αcompetent cells, and the next day a single clone was picked forsequencing. The plasmid which were verified correct by sequencing wererespectively named as pBD-H1N1_(SC09)-PB2 plasmid, pBD-H1N1_(SC09)-PB1plasmid, pBD-H1N1_(SC09)-PA plasmid, pBD-H1N1_(SC09)-NP plasmid,pBD-H1N1_(SC09)-HA plasmid, pBD-H1N1_(SC09)-NA plasmid,pBD-H1N1_(SC09)-M plasmid and pBD-H1N1_(SC09)-NS plasmid for subsequentexperiments.

TABLE 10 The primers for constructing H1N1SC09  pBD eight plasmidsprimer name primer sequence (5′-3′) pBD-up, GGCCGGCATGGTCCCAGCCTCCTCSEQ ID NO: 95 GC pBD-down, AATAACCCGGCGGCCCAAAATGCC SEQ ID NO: 96 GACTCGpBD-PB2-F, GGGACCATGCCGGCCAGCAAAAG SEQ ID NO: 97 CAGGTCAAATATATpBD-PB2-R, GGCCGCCGGGTTATTAGTAGAAAC SEQ ID NO: 98 AAGGTCGTTTTTAApBD-PB1-F, GGGACCATGCCGGCCAGCAAAAG SEQ ID NO: 99 CAGGCAAACCATTpBD-PB1-R, GGCCGCCGGGTTATTAGTAGAAAC SEQ ID NO: 100 AAGGCATTTTTTCApBD-PA-F, GGGACCATGCCGGCCAGCAAAAG SEQ ID NO: 101 CAGGTACTGATCCApBD-PA-R, GGCCGCCGGGTTATTAGTAGAAAC SEQ ID NO: 102 AAGGTACTTTTTTGGpBD-NP-F, GGGACCATGCCGGCCAGCAAAAG SEQ ID NO: 103 CAGGGTAGAT pBD-NP-R,GGCCGCCGGGTTATTAGTAGAAAC SEQ ID NO: 104 AAGGGTATTTTTC pBD-HA-F,GGGACCATGCCGGCCAGCAAAAG SEQ ID NO: 105 CAGGGGAAAA pBD-HA-R,GGCCGCCGGGTTATTAGTAGAAAC SEQ ID NO: 106 AAGGGTGT pBD-NA-F,GGGACCATGCCGGCCAGCAAAAG SEQ ID NO: 107 CAGGAGTTTAA pBD-NA-R,GGCCGCCGGGTTATTAGTAGAAAC SEQ ID NO: 108 AAGGAGTTT pBD-M-F,GGGACCATGCCGGCCAGCAAAAG SEQ ID NO: 109 CAGGTAGATA pBD-M-R,GGCCGCCGGGTTATTAGTAGAAAC SEQ ID NO: 110 AAGGTAGTTT pBD-NS-F,GGGACCATGCCGGCCAGCAAAAG SEQ ID NO: 111 CAGGGTGACAAAG pBD-NS-R,GGCCGCCGGGTTATTAGTAGAAAC SEQ ID NO: 112 AAGGGTGTTTTTTAT

The cell lines AKO, BKO, DKO constructed in Example 2 and wild type 293Tcell line were counted respectively and plated in a 6-well plate at4×10⁵/well. After cultured in an incubator at 37° C. for 20 h, thesystem of H1N1_(SC09) pBD 8 plasmids was transfected into a wild type293T cell line and a knockout cell line at 0.5 ug per plasmid; the cellsupernatant was collected at 0 h, 12 h, 24 h, 36 h, 48 h and 60 h aftertransfection, and the virus yield was determined by using an NP doubleantibody sandwich ELISA method: the 96-well ELISA plate was first coatedwith NP monoclonal antibody 2B8A11 at 1 ug/well (Wang Yadi, Wen Kun, QiuLiwen, etc., the establishment of ELISA capture method of influenza Avirus nucleocapsid protein and the clinical application thereof, 2009,Guangdong Medicine. 30(5): 703-705) at 4° C. overnight. The plate wasrinsed with 1×PBST washing solution for 3 times (5 minutes each time)and shaked, and then patted clean, added with 5% calf serum as ablocking solution, and blocked at 37° C. for 2 hr. The plate was againrinsed with 1×PBST washing solution for 3 times (5 minutes each time)and shaked, and then patted clean; the sample to be tested was added.The sample dilution used for the sample to be tested was 10% fetalbovine serum+0.1% TritonX-100 wherein the NP protein (NP proteinconstruction plasmid pET30a-NP is cited from Master's thesis: JiYuanyuan, the establishment of capture ELISA detection method of theequine influenza virus antigen and the primary application thereof [D].Chinese Academy of Agricultural Sciences, 2011) was used as a standardsample with a 2-fold dilution, and the collected cell supernatant wasdiluted 5-fold. Each sample was set with 3 gradients, 2 replicate wells.The plate was incubated at 37° C. for 2 h, rinsed with 1×PBST washingsolution for 3 times (5 minutes each time), and then patted clean; addedwith NP monoclonal antibody C16A15 strain (Wang Yadi, Wen Kun, QiuLiwen, etc., the establishment of ELISA capture method of influenza Avirus nucleocapsid protein and the clinical application thereof, 2009,Guangdong Medicine. 30(5): 703-705) by 1 ug/well, incubated at 37° C.for 1 h, discarded, rinsed with 1×PBST washing solution for 5 times (5minutes each time) and shaked. After the washing, 1:1 mixed AB colordeveloping solution (purchased from Beijing Taitianhe Biotech Co., Ltd.,cat # ME142) was added by 100 ul/well, and the color development wasperformed for 10 min. The color development was stopped by adding 2MH2504 at 50 ul/well, and OD450 nm was detected by using a biotech Ex150microplate reader. A standard curve was drawn by using the NP proteinstandard sample and the concentration of the sample to be tested wascalculated. The results showed that: compared to 293T cells, the amountof virus in the supernatant of AKO/BKO cells had a similar growth curveafter transfection, and almost no virus particles were detected in thesupernatant of DKO cells. This indicated that DKO cells did notsupported the replication and growth of virus, and the results wereshown in FIG. 12A. It was shown that the knockout of ANP32A or ANP32Balone did not affect the replication and growth of the virus.

The DKO cell lines constructed in Example 2 were counted respectivelyand plated in a 6-well plate at 3×10⁵/well. After incubated in anincubator at 37° C. for 20 h, transfection wells were set: 1 ug emptyvector PCAGGS-Flag, 1 ug PCAGGS-huANP32A, 1 ug PCAGGS-huANP32B, 0.5 ugPCAGGS-huANP32A+0.5 ug PCAGGS-huANP32B. 24 hours after transfection, thesystem of H1N1_(SC09) pBD 8 plasmids was transfected into cells ofdifferent treatment groups at 0.5 ug of each plasmid; aftertransfection, the cell supernatant was respectively collected at 0 h, 12h, 24 h, 36 h, 48 h and 60 h, and virus yield was determined by NPdouble antibody sandwich ELISA method, in which the specific steps weredescribed above. The results showed that: compared to the empty vectorof PCAGGS-Flag, the supplementation of huANP32A, the supplementation ofhuANP32B, and the simultaneous supplementation of both huANP32A andhuANP32B proteins all supported virus replication very well, and theresults were shown in FIG. 12B. In summary, the huANP32A or huANP32Bproteins was essential in the replication of H1N1_(SC09).

Example 7: The Experiment of H1N1/WSN Influenza Virus Infection

The cell lines AKO, BKO, DKO constructed in Example 2 and wild type 293Tcell line were counted respectively and plated in a 6-well plate at4×10⁵/well. After incubated in an incubator at 37° C. for 20 hours, thecells were infected with 0.01 MOI of WSN virus (Neumann G; et al.Generation of influenza A viruses entirely from cloned cDNAs.[J].Proceedings of the National Academy of Sciences of the United States ofAmerica, 1999, 96(16):9345-50); after 2 hours of virus adsorption, thevirus-infected solution was discarded and rinsed twice with 1×PBSbuffer, and then 2 ml of a cell maintenance solution containing 1%pancreatin (sigma)+1% double antibody (gibco)+0.5% fetal bovine serum(sigma) was added to each well, and cell infection supernatants weretaken at 0 h, 12 h, 24 h, 36 h, 48 h after infection and frozen at −80°C. for use. MDCK cells (canine kidney cell line, purchased from ChinaInstitute of Veterinary Drug Control) were plated in a 96-well plate at1.5×10⁴/well, and the above-obtained supernatant was 10-fold dilutedwith culture solution DMEM (hyclone) and then added into a 96-well plateat 100 ul/well with 8 replicates per gradient. After 2 hours of virusadsorption, the virus infection solution was discarded and the wellswere rinsed twice by 1×PBS buffer, then 100 ul of cell maintenancesolution containing 2% fetal bovine serum (sigma)+1% double antibody(gibco)+1% pancreatin (sigma) was added into each well; after 48 hours,the cell lesion was observed and counted; virus TCID50 at different timepoints of different treatment groups was calculated according to aReed-Muench method, and finally, the virus growth curve was drawn byGraphpad prism 5 software. The result showed that: the virus growthcurves of AKO and BKO cells were consistent with that of wild-type 293Tcells, whereas DKO cells hardly supported virus growth. The result wasshown in FIG. 13A.

Double-knockout cell lines (DKO) were plated in a 6-well plate at4×10⁵/well; 20 hours later, four transfection groups were set:PCAGGS-huANP32A (1 ug) plasmid, PCAGGS-huANP32B (1 ug) plasmid,PCAGGS-huANP32A+ PCAGGS-huANP32B (0.5 ug+0.5 ug), and PCAGGS-Flag emptyvector (1 ug). After 24 hours of transfection, cells of differenttreatment groups were infected with 0.01 MOI of WSN virus; after 2 hoursof virus adsorption, the virus infection solution was discarded and thewells were rinsed twice with 1×PBS buffer, and then 2 ml of a cellmaintenance solution containing 1% pancreatin (sigma)+1% double antibody(gibco)+0.5% fetal bovine serum (sigma) was added to each well, andvirus infection supernatants were taken at 0 h, 12 h, 24 h, 36 h, 48 hafter infection and frozen at −80° C. for use. MDCK cells were plated ina 96-well plate at 1.5×10⁴/well, and the above-obtained supernatant was10-fold diluted and then added into a 96-well plate at 100 ul/well with8 replicates per gradient. After 2 hours of virus adsorption, the virusinfection solution was discarded and rinsed twice by 1×PBS buffer, thenthe cell maintenance solution was added; after 48 hours, the cell lesionwas observed and counted; virus TCID50 at different time points ofdifferent treatment groups was calculated according to a Reed-Muenchmethod, and finally, the virus growth curve was drawn by Graphpad prism5 software. The result showed that: compared with the empty vector,supplementation of huANP32A or huANP32B alone can restore the growth ofvirus in DKO cells, which was consistent with the virus growth curves ofsupplementation of both huANP32A and huANP32B. The result was shown inFIG. 13B.

It was shown that the knockout of huANP32A or huANP32B alone did notaffect the replication and growth of the virus, and that huANP32A andhuANP32B had a functional compensation effect on the replication andgrowth of influenza virus.

Example 8: Influence of ANP32A and ANP32B Proteins on PolymeraseReplication after Mutation of Homologous or Heterologous VirusConstruction of Point Mutation Vector of PB2 Gene of H7N9 SubtypeInfluenza Virus

The analysis of some key amino acid sites on PB2 gene of a human-derivedH7N9 isolated strain showed that compared with an avian-derivedisolates, the human-derived isolates had some reported point mutationsrelated to host adaptability, such as A588V, Q591K, Q591R, V598I, E627K,D701N and the like (Hu et al., 2017, PB2 substitutions V598T/I increasethe virulence of H7N9 influenza A virus in mammals. Virology. 501,92-101.; Mok et al., 2014, Amino acid substitutions in polymerase basicprotein 2 gene contribute to the pathogenicity of the novel A/H7N9influenza virus in mammalian hosts. Journal of virology. 88(6),3568-3576; Xiao et al., 2016, PB2-588 V promotes the mammalianadaptation of H10N8, H7N9 and H9N2 avian influenza viruses. Sci Rep. 6,19474.; Yamayoshi et al., 2015, Amino acids substitutions in the PB2protein of H7N9 influenza A viruses are important for virulence inmammalian hosts[J]. Sci Rep, 2015, 5:8039.; Zhang et al., 2014, The PB2E627K mutation contributes to the high polymerase activity and enhancedreplication of H7N9 influenza virus. J Gen Virol. 95(Pt 4), 779-786.),that is, after the point mutations of A588V, Q591K, Q591R, V598I, E627Kor D701N were performed on the avian-derived influenza strain, thestrain became adaptive to a human body.

(1) Mutant PB2 Gene and Plasmid Construction

We performed a single point mutation at the above six positions on PB2of avian H7N9 influenza A/chicken/Zhejiang/DTID-ZJU01/2013(H7N9_(ZJ13));the mutation primers were shown in Table 11 (the underlined parts weremutant bases); the avian H7N9 (H7N9_(ZJ13)) PB2 plasmid was used as atemplate, and KOD-FX Neo high-efficiency DNA polymerase was used foramplification; the obtained PCR product was digested with Dpn I for 30minutes in a constant temperature water bath at 37° C., then 5 ul of thedigested product was transformed into 20 ul of DH5α competent cells; thenext day, a single clone was picked for sequencing, and a large amountof plasmid which was verified correct by sequencing was extracted forlater use. PB2(A588V), PB2(Q591K), PB2(Q591R), PB2(V598I), PB2(E627K)and PB2(D701N) mutant genes were obtained, respectively.

TABLE 11 Mutation primers primer name primer sequence ZJ13-PB2 A588V-S,CTAAAGCTGTCAGAGGCCAATAT SEQ ID NO: 113 AGTG ZJ13-PB2 A588V-A,TATTGGCCTCTGACAGCTTTAGG SEQ ID NO: 114 CACT ZJ13-PB2 Q591K-S,TGCCAGAGGCAAATATAGTGGG SEQ ID NO: 115 TTCGTG ZJ13-PB2 Q591K-A,CCCACTATATTTGCCTCTGGCAG SEQ ID NO: 116 CTTTA ZJ13-PB2 Q591R-S,TGCCAGAGGCAGATATAGTGGG SEQ ID NO: 117 TTCGTG ZJ13-PB2 Q591R-A,CCCACTATATCTGCCTCTGGCAG SEQ ID NO: 118 CTTTA ZJ13-PB2 V598I-S,AGTGGGTTCGTGAGGATTCTATT SEQ ID NO: 119 CCAACAGATG ZJ13-PB2 V5981-A,CATCTGTTGGAATAGAATCCTC SEQ ID NO: 120 ACGAACCCACT ZJ13-PB2 E627K-S,GCAGCCCCGCCGAAGCAGAGTA SEQ ID NO: 121 GGATGCA ZJ13-PB2 E627K-A,ATCCTACTCTGCTTCGGCGGGGC SEQ ID NO: 122 TGCTGCA ZJ13-PB2 D701N-S,GGGCAAAGAAAATAAAAGATAT SEQ ID NO: 123 GGGCCA ZJ13-PB2 D701N-A,CCATATCTTTTATTTTCTTTGCCC SEQ ID NO: 124 AGAATC(2) Influence of huANP32A and huANP32B on Polymerase Replication

Double-knockout cell lines (DKO) were plated in a 12-well plate at3×10⁵/well; after 20 hours, the plasmids of PCAGGS-chANP32A,PCAGGS-huANP32A, pCAGGS-huANP32B constructed in Example 1 and the emptyvector PCAGGS-Flag were respectively co-transfected with the plasmids ofH7N9_(ZJ13) polymerase reporter system. The transfection system was: PB1plasmid (80 ng), PB2 plasmid (80 ng, that is, PB2(A588V), PB2(Q591K),PB2(Q591R), PB2(V598I), PB2(E627K) and PB2(D701N) were respectivelyused), PA plasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80ng), pRL-TK plasmid (10 ng), and ANP32A protein plasmid (20 ng), and theempty vector PCAGGS-Flag (20 ng) was set as a negative control, and eachgroup was provided with triplicate wells.24 hours after transfection,the cells were lysed for detecting the activity of polymerase, and theresult showed that: compared with the empty vector, chANP32A, huANP32Aand huANP32B can effectively promote the activity of polymerase withpoint mutations of A588V, Q591K, Q591R, V598I, D701N and E627K; in theabsence of huANP32A and huANP32B, none of these point mutations allowedthe polymerase to acquire the ability to replicate on 293T; theseresults indicated that: for the replication in humans of thehuman-derived influenza strain or mutant strain which was obtained bymutation of avian-derived influenza strain to adapt to the human body,huANP32A or huANP32B was a prerequisite for the replication of H7N9polymerase in 293T. The result was shown in FIG. 14.

Example 9: Determination of Functional Domain of ANP32 Protein

According to the results in Example 5, it was shown that chANP32Bprotein did not support the activity of polymerase.

By aligning the protein sequences of chANP32B, huANP32B and pgANP32B, itwas found that there are differences in the sequence of ANP32B (FIG.15A). According to the UniProtKB database, the functional domains ofhuANP32B protein were displayed: 1-41aa was the LRR1 region; 42-110aawas the LRR2, 3 &4 region; 111-161aa was the LRRCT region; and 162-251aawas the LCAR region. According to the functional domain of the huANP32Bprotein, the huANP32B gene fragment and the chANP32B gene fragment werereplaced, and the replacement strategy of the gene fragment was shown inFIG. 15B.

Firstly, the huANP32B gene sequence was divided into two fragments:1-161aa and 162-262aa; by using a homologous recombination PCR method,primers were designed to replace the corresponding fragment with achANP32B fragment to construct a recombinant plasmid.

For example, the replacement of 1-161aa fragment was performed asfollows: using a PCAGGS-chANP32B plasmid as a template, aPCAGGS-chANP32BΔ1-161 gene (SEQ ID NO:125 and SEQ ID NO:126 as primers)lacking the 1-161aa gene fragment was amplified by KOD-FX Neohigh-efficiency DNA polymerase; then by using a PCAGGS-huANP32B plasmidas a template, a huANP32B (1-161) fragment was amplified (SEQ ID NO:127and SEQ ID NO:128 as primers), wherein both ends of the primersamplifying the huANP32B (1-161) fragment respectively contain 15 bpbases which are the same as the left and right arms of thePCAGGS-chANP32BΔ1-161 gene. After the fragments were amplified andrecovered, the two fragments were ligated by In-Fusion ligase (purchasedfrom Clontech) according to the instructions, and then transformed intoDH5α competent cells. The next day, a single clone was picked andsequenced, and the plasmid which was verified correct by sequencing wasnamed as chANP32B(162-262) and was extracted in large-scale for lateruse. The primers were shown in Table 12.

The chANP32B(1-161) recombinant plasmid was constructed according to themethod described above. By using a PCAGGS-chANP32B plasmid as atemplate, a PCAGGS-chANP32BΔ162-262 gene lacking the 162-262aa genefragment was amplified by KOD-FX Neo high-efficiency DNA polymerase (ca#KFX-201, purchased from Toyobo) (SEQ ID NO:129 and SEQ ID NO:130 asprimers); then by using a PCAGGS-huANP32B plasmid as a template, ahuANP32B(162-262) fragment was amplified(SEQ ID NO:131 and SEQ ID NO:132as primers), wherein both ends of the primers used for amplifying thehuANP32B (162-262) fragment respectively contain 15 bp bases which arethe same as the left and right arms of the PCAGGS-chANP32BΔ162-262 gene.After the fragments were amplified and recovered, the two fragments wereligated by In-Fusion ligase (purchased from Clontech) according to theinstructions, and then transformed into DH5α competent cells. The nextday, a single clone was picked and sequenced, and the plasmid which wasverified correct by sequencing was named as chANP32B(1-161) and wasextracted in large-scale for later use. The primers were shown in Table12.

TABLE 12 primers primer name primer sequence pCAGGS Vector_up,GCGGCCGCGAGCTCGAATTCTTTGCCAA SEQ ID NO: 125 AA pCAGGS_ch32BGAGGCAGATGGGGATGGACTGGAAGAC (162-262)-down GAG SEQ ID NO: 126hu32B(1-161)_F, GAATTGTGCGGCCGCATGGACATGAAG SEQ ID NO: 127 AGGAGGATCCAhu32B(1-161)_R, ATCCCCATCTGCCTCGGCATCTGAGTCA SEQ ID NO: 128 GGTGCTTCCTpCAGGS_ch32B AGGGTCTGAGTCAGGGGCTTCCTGCTCA (1-161)-vector up TCSEQ ID NO: 129 pCAGGS GGCAGCGGAGACTACAAGGATGACGAT Vector_down, GACSEQ ID NO: 130 hu32B(162-262)_F, CCTGACTCAGACCCTGAGGTGGATGGTGSEQ ID NO: 131 TGGATGAAGA hu32B(162-262)_R.GTAGTCTCCGCTGCCATCATCTTCTCCTT SEQ ID NO: 132 CATCATCTG

Double-knockout cell lines (DKO) were plated in a 12-well plate at3×10⁵/well; after 20 hours, the plasmids of PCAGGS-huANP32B,PCAGGS-chANP32B, PCAGGS-chANP32B(1-161) and PCAGGS-chANP32B(162-262)were respectively co-transfected with the H1N1_(SC09) polymerasereporter system into the DKO cell line. The transfection system was: PB1plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid(160 ng), pMD18T-vLuc plasmid (80 ng), pRL-TK plasmid (10 ng) and ANP32Aprotein plasmid (20 ng), and each group was provided with triplicatewells. 24 hours after transfection, the cells were lysed and theactivity of polymerase was detected; the result was shown in FIG. 15C:the ability of chANP32B(1-161) to support H1N1_(SC09) polymeraseactivity was lost, while chANP32B(162-262) retained the ability tosupport H1N1_(SC09) polymerase activity. Therefore, the key region ofthe protein ANP32B that supports polymerase activity is located within1-161aa.

The homologous recombination PCR was performed as described above; byusing huANP32B as a backbone, chANP32B 1-161aa was further divided intothree regions of 1-41aa, 42-110aa and 111-161aa to replace thecorresponding fragments of PCAGGS-huANP32B, to construct the recombinantplasmid PCAGGS-chANP32B(1-41) (using plasmid PCAGGS-huANP32B as atemplate, using the primer pair SEQ ID NO:133 and SEQ ID NO: 134 toamplify the PCAGGS-hu32A(1-41) gene fragment; using the PCAGGS-chANP32Bplasmid as a template, using the primer pair SEQ ID NO: 135 and SEQ IDNO: 136 to amplify the chANP32B (1-41) gene fragment), PCAGGS-chANP32B(42-110) (using the PCAGGS-huANP32B plasmid as a template, using theprimer pair SEQ ID NO: 137 and SEQ ID NO: 138 to amplify thePCAGGS-hu32BA(42-110) gene fragment; using the PCAGGS-chANP32B plasmidas a template, using the primer pair SEQ ID NO: 139 and SEQ ID NO: 140to amplify the chANP32B (42-110) gene fragment) and PCAGGS-chANP32B(111-161) (using the PCAGGS-huANP32B plasmid as a template, using primerpair SEQ ID NO: 141 and SEQ ID NO: 142 to amplify thePCAGGS-hu32BA(111-161) gene fragment; using the PCAGGS-chANP32B plasmidas a template; using the primer pair SEQ ID NO: 143 and SEQ ID NO: 144to amplify the chANP32B (111-161) gene fragment). The primers were shownin Table 13.

TABLE 13 primers primer name primer sequence pCAGGS Vector_up,GCGGCCGCGAGCTCGAATTCTTTGCCAA SEQ ID NO: 133 pCAGGS_hu32BGTGAACTTAGAGTTCCTCAGTTTAATAAA (42-262)-down T SEQ ID NO: 134ch32B(1-41)_F, GAATTGTGCGGCCGCATGGAGATGAAAA SEQ ID NO: 135 AGCGGCTCACch32B(1-41)_R, GAACTCTAAGTTCACAAAATCTGAAGAGA SEQ ID NO: 136 GCCCAACGApCAGGS_hu32B AAATTCAGCTGTTAAGCCCTCAATTTTTCC (1-41)_up, SEQ ID NO: 137pCAGGS_hu32B AAGTTAGAATGTCTGAAAAGCCTGGACCT (111-262)_down CSEQ ID NO: 138 ch32B(42-110)_F, TTAACAGCTGAATTTGAGAACCTGGAGTTSEQ ID NO: 139 CCTCAGCAT ch32B(42-110)_R, CAGACATTCTAACTTTTTCAAGGGTTCCASEQ ID NO: 140 GGGTATTGA pCAGGS_hu32B TTTCAAAGGTTCCAAGGTGCTGATATCTTT(1-110)-up SEQ ID ID: 141 pCAGGS_hu32B CTCCTCCTCTTCATCCACACCATCCACCTC(162-262)_down SEQ ID NO: 142 ch32B(111-161)_F,TTGGAACCTTTGAAAAAGTTGCCAAACCT SEQ ID NO: 143 GCATAGTCT ch32B(111-161)_R,CACACCATCCACCTCAGGGTCTGAGTCAG SEQ ID NO: 144 GGGCTTCCT

Double-knockout cell lines (DKO) were plated in a 12-well plate at3×10⁵/well; after 20 hours, the plasmids of PCAGGS-huANP32B,PCAGGS-chANP32B, PCAGGS-chANP32B (1-41), PCAGGS-chANP32B(42-110) andPCAGGS-chANP32B(111-161) were respectively co-transfected with theH1N1_(SC09) polymerase reporter system into the DKO cell line. Thetransfection system was: PB1 plasmid (80 ng), PB2 plasmid (80 ng), PAplasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80 ng),pRL-TK plasmid (10 ng) and ANP32A protein plasmid (20 ng), and eachgroup was provided with triplicate wells. 24 hours after transfection,the cells were lysed as described in Example 3 and the activity ofpolymerase was detected; the result was shown in FIG. 15D: therecombinant plasmid PCAGGS-chANP32B(111-161) lost the ability to supportthe activity of H1N1_(SC09) polymerase, while the recombinant plasmidsPCAGGS-chANP32B(1-41) and PCAGGS-chANP32B(42-110) still maintained thesupport for the activity of H1N1_(SC09) polymerase.

The homologous recombination PCR was performed as described above, andthe corresponding fragment of PCAGGS-chANP32B was replaced with thecorresponding 111-161aa sequence of PCAGGS-huANP32B (using thePCAGGS-huANP32B plasmid as a template and using the primer pair of SEQID NO: 145 and SEQ ID NO:146 to amplify the PCAGGS-ch32BA(111-161) genefragment; using the PCAGGS-huANP32B plasmid as a template and using theprimer pair of SEQ ID NO:147 and SEQ ID NO:148 to amplify the huANP32B(111-162) gene fragment), to construct the recombinant plasmidPCAGGS-chANP32B(hu111-161); the primers were shown in Table 14. DKO cellline was co-transfected with the H1N1_(SC09) polymerase reporter systemaccording to the above system, and the polymerase activity was detectedas described in Example 3; the result was shown in FIG. 15D (column ofchANP32B(hu111-161)), indicating that chANP32B(hu111-161) acquired theability to support the activity of H1N1_(SC09) polymerase, which furtherindicated that the key region of the ANP32B protein to supportpolymerase activity was located within 111-161aa.

TABLE 14 primers primer name primer sequence pCAGGS_ch32BCTTTTTCAAGGGTTCCAGGGTATTGATGTC (1-110)_up SEQ ID NO: 145 pCAGGS_ch32BGAGGCAGATGGGGATGGACTGGAAGACG (162-262)_down AG SEQ ID NO: 146hu32B(111-161)_F GAACCCTTGAAAAAGTTAGAATGTCTGAA SEQ ID NO: 147 AAGCCTGGAhu32B(111-161)_R ATCCCCATCTGCCTCGGCATCTGAGTCAG SEQ ID NO: 148 GTGCTTCCT

To further identify the key regions, alignment of the protein sequencesof chANP32B, huANP32B and pgANP32B revealed that amino acids in the111-161aa region of huANP32B and pgANP32B proteins were relativelyconserved, while there were differences of mainly eight amino acidsbetween chANP32B and the above two proteins (positions 113, 116, 127,129, 130, 137, 150 and 160, respectively), as shown in FIG. 16A. Mutantprimers were designed for these 8 amino acids in the huANP32B sequence(see Table 15 for the primers, and the mutant bases were underlined); byusing the homologous recombination method as described above, thefollowing point mutants were respectively constructed by using KOD FXNeo polymerase with PCAGGS-huANP32B plasmid as template: huANP32B E113P(using primer pair of SEQ ID NO: 149 and SEQ ID NO: 150), K116H (usingprimer pair of SEQ ID NO: 151 and SEQ ID NO: 152), N127M (using primerpair of SEQ ID NO: 153 and SEQ ID NO: 154), N129I/D130N (using primerpair SEQ ID NO: 155 and SEQ ID NO: 156), K137T (using primer pair of SEQID NO: 157 and SEQ ID NO: 158), R150A (using primer pair of SEQ ID NO:159 and SEQ ID NO: 160), A160P (using primer pair of SEQ ID NO: 161 andSEQ ID NO: 162), which were respectively named as PCAGGS-huANP32B E113P,PCAGGS-huANP32B K116H, PCAGGS-huANP32B N127M, PCAGGS-huANP32BN129I/D130N, PCAGGS-huANP32B K137T, PCAGGS-huANP32B R150A,PCAGGS-huANP32B A160P, for use in the next step of transfection afterconfirming by sequencing.

TABLE 15 primers for amino acid mutation primer nameprimer sequence (5′-3′) huB_E113P_F CCTTTGAAAAAGTTACCCTGTCTGAAAAGCCSEQ ID NO: 149 TG huB_E113P_R CAGGCTTTTCAGACAGGGTAACTTTTTCAAASEQ ID NO: 150 GG huB_K116H_F AAGTTAGAATGTCTGCACAGCCTGGACCTCTSEQ ID NO: 151 TT huB_K116H_R AAAGAGGTCCAGGCTGTGCAGACATTCTAACSEQ ID NO: 152 TT huB_N127M_F AACTGTGAGGTTACCATGCTGAATGACTACCSEQ ID NO: 153 GA huB_N127M_R TCGGTAGTCATTCAGCATGGTAACCTCACAGTSEQ ID NO: 154 T huB_N129I/ GAGGTTACCAACCTGATTAACTACCGAGAGA D130N_FGTGTC SEQ ID NO: 155 huB_N129I/ GACACTCTCTCGGTAGTTAATCAGGTTGGTAA D130N_RCCTC SEQ ID NO: 156 huB_K137T_F CGAGAGAGTGTCTTCACCCTCCTGCCCCAGCSEQ ID NO: 157 TT huB_K137T_R AAGCTGGGGCAGGAGGGTGAAGACACTCTCSEQ ID NO: 158 TCG huB_R150A_F TTGGATGGCTATGACGCTGAGGACCAGGAAGSEQ ID NO: 159 CA huB_R150A_R TGCTTCCTGGTCCTCAGCGTCATAGCCATCCASEQ ID NO: 160 A huB_A160P_F GCACCTGACTCAGATCCGGAGGTGGATGGTGSEQ ID NO: 161 TG huB_A160P_R CACACCATCCACCTCCGGATCTGAGTCAGGTSEQ ID NO: 162 GC

Double-knockout cell lines (DKO) were plated in a 12-well plate at3×10⁵/well; after 20 hours, the point mutant plasmids of PCAGGS-Flag,PCAGGS-huANP32B, PCAGGS-chANP32B and PCAGGS-huANP32B, namely,PCAGGS-huANP32B E113P, PCAGGS-huANP32B K116H, PCAGGS-huANP32B N127M,PCAGGS-huANP32B N291/D130N, PCAGGS-huANP32B K137T, PCAGGS-huANP32BR150A, PCAGGS-huANP32B A160P were respectively co-transfected with theH1N1_(SC09) polymerase reporter system into the DKO cell line. Thetransfection system was: PB1 plasmid (80 ng), PB2 plasmid (80 ng), PAplasmid (40 ng), NP plasmid (160 ng), pMD18T-vLuc plasmid (80 ng),pRL-TK plasmid (10 ng) and ANP32A mutant protein plasmid (20 ng), andeach group was provided with triplicate wells. 24 hours aftertransfection, the cells were lysed as described in Example 3 and thepolymerase activity were detected; the result was shown in FIG. 16B:compared to huANP32B, chANP32B and huANP32B N129I/D130N completely lostsupport for the activity of H1N1_(SC09) polymerase, while the pointmutants of huANP32B E113P, huANP32B K116H, huANP32B N127M, huANP32BK137T, huANP32B R150A and huANP32B A160P still retained support for theactivity of H1N1_(SC09) polymerase.

For the two sites 129/130, single point mutations of huANP32B N129I(using primer pair of SEQ ID NO:163 and SEQ ID NO: 164) and D130N (usingprimer pair of SEQ ID NO: 165 and SEQ ID NO: 166) were designed (seeTable 16 for primers, and the mutated bases were underlined), and theresulting plasmids were named as PCAGGS-huANP32B N129I andPCAGGS-huANP32B D130N, wherein the PCAGGS-huANP32B plasmid was used as atemplate and the procedure was as described in this Example for thepoint mutation of 8 amino acids in the huANP32B sequence. Afterverification by sequencing, the plasmids were extracted in large amountfor further transfection.

TABLE 16 primers for single point mutation huANP32B N129I and D130Nprimer name primer sequence (5′-3′) huB_N129I_FGAGGTTACCAACCTGATTGACTACCGAGAGAGT SEQ ID NO: 163 huB_N129I_RACTCTCTCGGTAGTCAATCAGGTTGGTAACCTC SEQ ID NO: 164 huB_D130N_FGTTACCAACCTGAATAACTACCGAGAGAGTGTC SEQ ID NO: 165 huB_D130N_RGACACTCTCTCGGTAGTTATTCAGGTTGGTAAC SEQ ID NO: 166

Double-knockout cell line (DKO) was plated in a 12-well plate at3×10⁵/well and transfected as described above after 20 h, and the resultshowed that: compared to huANP32B, huANP32B N129I almost lost supportfor H1N1_(SC09) polymerase activity, while the support of huANP32B D130Nfor H1N1_(SC09) polymerase activity was reduced by about 5 times. Thisshowed that the two sites of 129/130 were important for the activity ofthe ANP32 protein. The result was shown in FIG. 17.

Double-knockout cell lines (DKO) were plated in a 12-well plate at3×10⁵/well; after 20 hours, the point mutation plasmids of PCAGGS-Flagempty vector, PCAGGS-huANP32B and PCAGGS-huANP32B were respectivelyco-transfected with the H7N9_(AH13) polymerase reporter system into theDKO cell line. The transfection system was: PB1 plasmid (80 ng), PB2plasmid (80 ng), PA plasmid (40 ng), NP plasmid (160 ng), pMD18T-vLucplasmid (80 ng), pRL-TK plasmid (10 ng) and ANP32A mutant proteinplasmid (20 ng), and each group was provided with triplicate wells.24hours after transfection, the cells were lysed as described in Example 3and the polymerase activity were detected; the result showed that:compared to huANP32B, huANP32B N129I and huANP32B N129I/D130N completelylost support for the activity of H7N9_(AH13) polymerase, while the pointmutants of huANP32B K116H, huANP32B N127M, huANP32B R150A and huANP32BA160P still retained support for the activity of H7N9_(AH13) polymerase,and the ability of huANP32B E113P, huANP32B D130N and huANP32B K137T tosupport the activity of H7N9_(AH13) polymerase was reduced by about 3-8times. The result was shown in FIG. 18.

According to the screening results of huANP32B point mutation, huANP32Awas also subjected to the point mutant construction of N129I (usingprimer pair of SEQ ID NO: 167 and SEQ ID NO: 168), D130N (using primerpair of SEQ ID NO: 169 and SEQ ID NO: 170) and ND129/130IN (using primerpair of SEQ ID NO: 171 and SEQ ID NO: 172) (see Table 17 for primers,and mutated bases were underlined) by using overlapping PCR with thePCAGGS-huANP32A plasmid as a template, wherein the procedure is asdescribed in the construction of a point mutant of 8 amino acids on thehuANP32B sequence. As described above, the obtained plasmids were namedas PCAGGS-huANP32A N129I, PCAGGS-huANP32A D130N and PCAGGS-huANP32AN129I/D130N. After verification by sequencing, the plasmids wereextracted in large amount for further transfection.

TABLE 17 Primers for point mutations of N129I, D130N,N129I/D130N on huANP32A primer name primer sequence (5′-3′) huA_N129I_FGAGGTAACCAACCTGATTGACTACCGAGAAA SEQ ID NO: 167 AT huA_N129I_RATTTTCTCGGTAGTCAATCAGGTTGGTTACCT SEQ ID NO: 168 C huA_D130N_FGTAACCAACCTGAACAACTACCGAGAAAATG SEQ ID NO: 169 TG huA_D130N_RCACATTTTCTCGGTAGTTGTTCAGGTTGGTTA SEQ ID NO: 170 C huA_N129I/GAGGTAACCAACCTGATTAACTACCGAGAAA D130N_F ATGTG SEQ ID NO: 171 huA_N129I/CACATTTTCTCGGTAGTTAATCAGGTTGGTTA D130N_R CCTC SEQ ID NO: 172

Double-knockout cell line (DKO) was plated in a 12-well plate at3×10⁵/well and co-transfected with the H1N1_(SC09) polymerase reportersystem as described above after 20 h, and the result showed that:compared to huANP32A, huANP32A N129I/D130N completely lost support forH1N1_(SC09) polymerase activity, huANP32A N129I almost lost support forH1N1_(SC09) polymerase activity, while the ability of huANP32B D130N tosupport H1N1_(SC09) polymerase activity was reduced by more than 100times, as shown in FIG. 19.

Double-knockout cell line (DKO) was plated in a 12-well plate at3×10⁵/well and after 20 h was co-transfected with the H7N9_(SC09)polymerase reporter system as described above, and the result showedthat: huANP32A N129I, huANP32A D130N and huANP32A N129I/D130N completelylost support for H7N9_(AH13) polymerase activity as compared withhuANP32A. The result was shown in FIG. 20.

According to the screening results of huANP32B point mutation, chANP32Awas subjected to the point mutations of N129I (using primer pair of SEQID NO: 173 and SEQ ID NO: 174), D130N (using primer pair of SEQ ID NO:175 and SEQ ID NO: 176) and N129I/D130N (using primer pair of SEQ ID NO:177 and SEQ ID NO: 178) by overlapping PCR using PCAGGS-chANP32A plasmidas a template; at the same time, chANP32B was subjected to the pointmutations of I129N (using primer pair of SEQ ID NO: 179 and SEQ ID NO:180), N130D (using primer pair of SEQ ID NO: 181 and SEQ ID NO: 182) andI129N/N130D (using primer pair of SEQ ID NO: 183 and SEQ ID NO: 184) byusing PCAGGS-chANP32B plasmid as a template (see Table 18 for primers,and the mutated bases were underlined.) After verification bysequencing, the plasmids were extracted in large amount for furthertransfection.

TABLE 18 Primers for point mutations of chANP32A and chANP32Bprimer name primer sequence (5′-3′) chA_N129I_FGAGGTAACCAACTTGATTGATTATAGAGAAA SEQ ID NO: 173 AC chA_N129I_RGTTTTCTCTATAATCAATCAAGTTGGTTACCTC SEQ ID NO: 174 chA_D130N_FGTAACCAACTTGAATAACTATAGAGAAAACG SEQ ID NO: 175 TA chA_D130N_RTACGTTTTCTCTATAGTTATTCAAGTTGGTTAC SEQ ID NO: 176 chA_ND129/GAGGTAACCAACTTGATTAACTATAGAGAAA 130IN_F ACGTA SEQ ID NO: 177 chA_ND129/TACGTTTTCTCTATAGTTAATCAAGTTGGTTAC 130IN_R CTC SEQ ID NO: 178 chB_I129N_FGAGGTGACGATGCTCAATAACTACCGGGAGA SEQ ID NO: 179 GT chB_I129N_RACTCTCCCGGTAGTTATTGAGCATCGTCACCT SEQ ID NO: 180 C chB_N130D_FGTGACGATGCTCATCGACTACCGGGAGAGTG SEQ ID NO: 181 TG chB_N130D_RCACACTCTCCCGGTAGTCGATGAGCATCGTC SEQ ID NO: 182 AC chB_IN129/GAGGTGACGATGCTCAATGACTACCGGGAGA 130ND_F GTGTG SEQ ID NO: 183 chB_IN129/CACACTCTCCCGGTAGTCATTGAGCATCGTCA 130ND_R CCTC SEQ ID NO: 184

Double-knockout cell line (DKO) was plated in a 12-well plate at3×10⁵/well and co-transfected with the H7N9_(AH13) polymerase reportersystem as described above after 20 h, and the result showed that:compared with chANP32A, chANP32A N129I/D130N lost the support forH7N9_(AH13) polymerase activity, the ability of chANP32A N129I tosupport H7N9_(AH13) polymerase activity was decreased by more than 100times, the ability of chANP32A D130N to support H7N9_(AH13) polymeraseactivity was decreased by about 5 times; compared with chANP32B,chANP32B I129N and chANP32B I129N/N130D had the ability to supportH7N9_(AH13) polymerase activity, while chANP32B N130D still did not havethe ability to support H7N9_(AH13) polymerase activity. The result wasshown in FIG. 21.

Example 10: Construction of the 129-Site Mutant of chANP32A Protein

Specifically, the primers for point mutation were shown in Table 19(mutated bases were underlined), using PCAGGS-chANP32A plasmid as atemplate, the following point mutants of chANP32A were constructed byKOD-FX Neo high-efficiency DNA polymerase: N129A (using primer pair SEQID NO: 185 and SEQ ID NO: 186), N129C (using primer pair SEQ ID NO: 187and SEQ ID NO: 188), N129D (using primer pair SEQ ID NO: 189 and SEQ IDNO: 190), N129E (using primer pair SEQ ID NO: 191 and SEQ ID NO: 192),N129F (using primer pair SEQ ID NO: 193 and SEQ ID NO: 194), N129G(using primer pair SEQ ID NO: 195 and SEQ ID NO: 196) N129H (usingprimer pair SEQ ID NO: 197 and SEQ ID NO: 198), N129K (using primer pairSEQ ID NO: 199 and SEQ ID NO: 200), N129L (using primer pair SEQ ID NO:201 and SEQ ID NO: 202), N129M (using primer pair SEQ ID NO: 203 and SEQID NO: 204), N129I (using primer pair SEQ ID NO: 173 and SEQ ID NO:174), N129P (using primer pair SEQ ID NO: 205 and SEQ ID NO: 206), N129Q(using primer pair SEQ ID NO: 207 and SEQ ID NO: 208), N129R (usingprimer pair SEQ ID NO: 209 and SEQ ID NO: 210), N129S (using primer pairSEQ ID NO: 211 and SEQ ID NO: 212), N129T (using primer pair SEQ ID NO:213 and SEQ ID NO: 214), N129V (using primer pair SEQ ID NO: 215 and SEQID NO: 216), N129W (using primer pair SEQ ID NO: 217 and SEQ ID NO:218), N129Y (using primer pair SEQ ID NO: 219 and SEQ ID NO: 220).

The obtained PCR product was digested with Dpn I in a 37° C. constanttemperature water bath for 30 minutes, and then 5 ul of the digestedproduct was taken and transformed into 20 ul of DH5α competent cells;the next day, a single clone was selected for sequencing, and theplasmid which was verified correct by sequencing was used for subsequenttransfection experiment.

TABLE 19 primers for point mutation primer name primer sequence (5′-3′)chA_N129A_F GAGGTAACCAACTTGGCAGATTATAGAGAAAAC SEQ ID NO: 185 chA_N129A_RGTTTTCTCTATAATCTGCCAAGTTGGTTACCTC SEQ ID NO: 186 chA_N129C_FGAGGTAACCAACTTGTGTGATTATAGAGAAAAC SEQ ID NO: 187 chA_N129C_RGTTTTCTCTATAATCACACAAGTTGGTTACCTC SEQ ID NO: 188 chA_N129D_FGAGGTAACCAACTTGGACGATTATAGAGAAAAC SEQ ID NO: 189 chA_N129D_RGTTTTCTCTATAATCGTCCAAGTTGGTTACCTC SEQ ID NO: 190 chA_N129E_FGAGGTAACCAACTTGGAAGATTATAGAGAAAAC SEQ ID NO: 191 chA_N129E_RGTTTTCTCTATAATCTTCCAAGTTGGTTACCTC SEQ ID NO: 192 chA_N129F_FGAGGTAACCAACTTGTTCGATTATAGAGAAAAC SEQ ID NO: 193 chA_N129F_RGTTTTCTCTATAATCGAACAAGTTGGTTACCTC SEQ ID NO: 194 chA_N129G_FGAGGTAACCAACTTGGGAGATTATAGAGAAAAC SEQ ID NO: 195 chA_N129G_RGTTTTCTCTATAATCTCCCAAGTTGGTTACCTC SEQ ID NO: 196 chA_N129H_FGAGGTAACCAACTTGCACGATTATAGAGAAAAC SEQ ID NO: 197 chA_N129H_RGTTTTCTCTATAATCGTGCAAGTTGGTTACCTC SEQ ID NO: 198 chA_N129K_FGAGGTAACCAACTTGAAGGATTATAGAGAAAAC SEQ ID NO: 199 chA_N129K_RGTTTTCTCTATAATCCTTCAAGTTGGTTACCTC SEQ ID NO: 200 chA_N129L_FGAGGTAACCAACTTGCTAGATTATAGAGAAAAC SEQ ID NO: 201 chA_N129L_RGTTTTCTCTATAATCTAGCAAGTTGGTTACCTC SEQ ID NO: 202 chA_N129M_FGAGGTAACCAACTTGATGGATTATAGAGAAAAC SEQ ID NO: 203 chA_N129M_RGTTTTCTCTATAATCCATCAAGTTGGTTACCTC SEQ ID NO: 204 chA_N129P_FGAGGTAACCAACTTGCCAGATTATAGAGAAAAC SEQ ID NO: 205 chA_N129P_RGTTTTCTCTATAATCTGGCAAGTTGGTTACCTC SEQ ID NO: 206 chA_N129Q_FGAGGTAACCAACTTGCAAGATTATAGAGAAAAC SEQ ID NO: 207 chA_N129Q_RGTTTTCTCTATAATCTTGCAAGTTGGTTACCTC SEQ ID NO: 208 chA_N129R_FGAGGTAACCAACTTGAGAGATTATAGAGAAAAC SEQ ID NO: 209 chA_N129R_RGTTTTCTCTATAATCTCTCAAGTTGGTTACCTC SEQ ID NO: 210 chA_N129S_FGAGGTAACCAACTTGAGCGATTATAGAGAAAAC SEQ ID NO: 211 chA_N129S_RGTTTTCTCTATAATCGCTCAAGTTGGTTACCTC SEQ ID NO: 212 chA_N129T_FGAGGTAACCAACTTGACAGATTATAGAGAAAAC SEQ ID NO: 213 chA_N129T_RGTTTTCTCTATAATCTGTCAAGTTGGTTACCTC SEQ ID NO: 214 chA_N129V_FGAGGTAACCAACTTGGTAGATTATAGAGAAAAC SEQ ID NO: 215 chA_N129V_RGTTTTCTCTATAATCTACCAAGTTGGTTACCTC SEQ ID NO: 216 chA_N129W_FGAGGTAACCAACTTGTGGGATTATAGAGAAAAC SEQ ID NO: 217 chA_N129W_RGTTTTCTCTATAATCCCACAAGTTGGTTACCTC SEQ ID NO: 218 chA_N129Y_FGAGGTAACCAACTTGTACGATTATAGAGAAAAC SEQ ID NO: 219 chA_N129Y_RGTTTTCTCTATAATCGTACAAGTTGGTTACCTC SEQ ID NO: 220

Example 11: Influence of the 129-Site Mutant of chANP32A Protein on theReplication of Influenza Virus H7N9_(ZJ13)

Double-knockout cell lines (DKO) were plated in a 12-well plate at3×10⁵/well; after 20 hours, the 129-site mutant of chANP32A constructedin Example 10 were respectively co-transfected with the 6 plasmids ofH7N9_(ZJ13) polymerase reporter system. The transfection system was: PB1(80 ng), PB2 (80 ng), PA (40 ng), NP (160 ng), pMD 18T-vLuc (80 ng),pRL-TK (10 ng) and the plasmid of ANP32 mutant protein (20 ng); and theempty vector (20 ng) was set as a negative control, chANP32A (20 ng) wasset as positive control, and each group was provided with triplicatewells.24 hours after transfection, the cells were lysed and the activityof polymerase was detected; The result showed that: compared withchANP32A, the two-point mutant of chANP32A N129I/D130N and thesingle-point mutants of chANP32A N129I, chANP32A N129R, chANP32A N129K,chANP32A N129D and chANP32A N129E did not have the ability to supportH7N9_(ZJ13) polymerase activity; chANP32A N129P, chANP32A N129Q,chANP32A N129G almost completely lost the ability to support H7N9_(ZJ13)polymerase activity, while chANP32A N129L, chANP32A N129F, chANP32AN129A, chANP32A N129M, chANP32A N129S, chANP32A N129T, chANP32A N129Cand chANP32A N129Y all supported H7N9_(ZJ13) polymerase activity; theability of chANP32A N129V, chANP32A N129W and chANP32A N129H to supportH7N9_(ZJ13) polymerase activity was reduced by approximately 100 times;the result was shown in FIG. 22.

Example 12: Influence of the 129-site mutant of chANP32A protein on thereplication of influenza virus H7N9_(AH13)

Double-knockout cell lines (DKO) were plated in a 12-well plate at3×10⁵/well; after 20 hours, the 129-site mutant of chANP32A constructedin Example 10 were co-transfected with the 6 plasmids of H7N9_(AH13)polymerase reporter system. The transfection system was: PB1 (80 ng),PB2 (80 ng), PA (40 ng), NP (160 ng), pMD 18T-vLuc (80 ng), pRL-TK (10ng) and the plasmid of ANP32 mutant protein (20 ng); and the emptyvector (20 ng) was set as a negative control, chANP32A (20 ng) was setas positive control, and each group was provided with triplicatewells.24 hours after transfection, the cells were lysed and the activityof polymerase was detected; The result showed that: compared withchANP32A, the two-point mutant of chANP32A N129I/D130N and thesingle-point mutants of chANP32A N129P, chANP32A N129R, chANP32A N129K,chANP32A N129Q, chANP32A N129D and chANP32A N129E did not have theability to support H7N9_(AH13) polymerase activity; chANP32A N129I haslittle ability to support H7N9_(AH13) polymerase activity; chANP32AN129F, chANP32A N129A, chANP32A N129M, chANP32A N129S, chANP32A N129G,chANP32A N129T, chANP32A N129C and chANP32A N129Y all supportedH7N9_(AH13) polymerase activity; the ability of chANP32A N129L andchANP32A N129W to support H7N9_(AH13) polymerase activity was reduced by3-10 times, and the ability of chANP32A N129V and chANP32A N129H tosupport H7N9_(AH13) polymerase activity was reduced by approximately20-100 times; the result was shown in FIG. 23.

Example 13: Influence of the 129-Site Mutant of chANP32A Protein on theReplication of Influenza Virus WSN

Double-knockout cell lines (DKO) were plated in a 12-well plate at3×10⁵/well; after 20 hours, the chANP32A 129-site mutant constructed inExample 10 were co-transfected with the 6 plasmids of WSN polymerasereporter system. The transfection system was: PB1 (80 ng), PB2 (80 ng),PA (40 ng), NP (160 ng), pMD 18T-vLuc (80 ng), pRL-TK (10 ng) and theplasmid of ANP32 mutant protein (20 ng); and the empty vector (20 ng)was set as a negative control, chANP32A (20 ng) was set as positivecontrol, and each group was provided with triplicate wells. 24 hoursafter transfection, the cells were lysed and the activity of polymerasewas detected; The result showed that: compared with chANP32A, thetwo-point mutant of chANP32A N129I/D130N and the single-point mutants ofchANP32A N129K and chANP32A N129D did not have the ability to supportWSN polymerase activity; while chANP32A N129F, chANP32A N129A, chANP32AN129M, chANP32A N129S, chANP32A N129G, chANP32A N129T and chANP32A N129Call supported WSN polymerase activity; the ability of chANP32A N129P,chANP32A N129I, chANP32A N129H, chANP32A N129R, chANP32A N129Q andchANP32A N129E to support WSN polymerase activity was reduced byapproximately 100 times; the ability of chANP32A N129L, chANP32A N129W,chANP32A N129Y and chANP32A N129V to support WSN polymerase activity wasreduced by approximately 5-20 times; the result was shown in FIG. 24.

Example 14: Construction of the 130-Site Mutant of chANP32A Protein

The primers for point mutation were shown in Table 20 (mutated baseswere underlined), using PCAGGS-chANP32A as a template, the followingpoint mutants of chANP32A were constructed by KOD-FX Neo high-efficiencyDNA polymerase amplification: N130A (using primer pair SEQ ID NO: 221and SEQ ID NO: 222), D130C (using primer pair SEQ ID NO: 223 and SEQ IDNO: 224), D130E (using primer pair SEQ ID NO: 225 and SEQ ID NO: 226),D130F (using primer pair SEQ ID NO: 227 and SEQ ID NO: 228), D130G(using primer pair SEQ ID NO: 229 and SEQ ID NO: 230), D130H (usingprimer pair SEQ ID NO: 231 and SEQ ID NO: 232), D130K (using primer pairSEQ ID NO: 233 and SEQ ID NO: 234), D130L (using primer pair SEQ ID NO:235 and SEQ ID NO: 236), D130M (using primer pair SEQ ID NO: 237 and SEQID NO: 238), D130N (using primer pair SEQ ID NO: 175 and SEQ ID NO:176), D130P (using primer pair SEQ ID NO: 239 and SEQ ID NO: 240), D130Q(using primer pair SEQ ID NO: 241 and SEQ ID NO: 242), D130R (usingprimer pair SEQ ID NO: 243 and SEQ ID NO: 244), D130S (using primer pairSEQ ID NO: 245 and SEQ ID NO: 246), D130T (using primer pair SEQ ID NO:247 and SEQ ID NO: 248), D130V (using primer pair SEQ ID NO: 249 and SEQID NO: 250), D130W (using primer pair SEQ ID NO: 251 and SEQ ID NO:252), D130Y (using primer pair SEQ ID NO: 253 and SEQ ID NO: 254), D1301(using primer pair SEQ ID NO: 255 and SEQ ID NO: 256).

The obtained PCR product was digested with Dpn I in a 37° C. constanttemperature water bath for 30 minutes, and then 5 ul of the digestedproduct was taken and transformed into 20 ul of DH5α competent cells;the next day, a single clone was picked for sequencing, and the plasmidswhich were verified correct by sequencing were used for subsequenttransfection experiment.

TABLE 20 primers of 130-site point mutation primer nameprimer sequence (5′-3′) chA D130A-F, SEQ ID NO: 221GTAACCAACTTGAATGCATAT AGAGAAAAC chA D130A-R, SEQ ID NO: 222CGTTTTCTCTATATGCATTCAA GTTGGTTACC chA D130C-F, SEQ ID NO: 223GTAACCAACTTGAATTGTTAT AGAGAAAAC chA D130C-R, SEQ ID NO: 224CGTTTTCTCTATAACAATTCA AGTTGGTTACCT chA D130E-F, SEQ ID NO: 225GTAACCAACTTGAATGAATAT AGAGAAAAC chA D130E-R, SEQ ID NO: 226CGTTTTCTCTATATTCATTCAA GTTGGTTACC chA D130F-F, SEQ ID NO: 227GTAACCAACTTGAATTTCTAT AGAGAAAAC chA D130F-R, SEQ ID NO: 228CGTTTTCTCTATAGAAATTCA AGTTGGTTACC chA D130G-F, SEQ ID NO: 229GTAACCAACTTGAATGGCTAT AGAGAAAAC chA D130G-R, SEQ ID NO: 230CGTTTTCTCTATAGCCATTCAA GTTGGTTACC chA D130H-F, SEQ ID NO: 231GTAACCAACTTGAATCACTAT AGAGAAAAC chA D130H-R, SEQ ID NO: 232CGTTTTCTCTATAGTGATTCAA GTTGGTTACC chA D130K-F, SEQ ID NO: 233GTAACCAACTTGAATAAGTAT AGAGAAAAC chA D130K-R, SEQ ID NO: 234CGTTTTCTCTATACTTATTCAA GTTGGTTACC chA D130L-F, SEQ ID NO: 235GTAACCAACTTGAATCTATAT AGAGAAAAC chA D130L-R, SEQ ID NO: 236CGTTTTCTCTATATAGATTCAA GTTGGTTACC chA D130M-F, SEQ ID NO: 237GTAACCAACTTGAATATGTAT AGAGAAAAC chA D130M-R, SEQ ID NO: 238CGTTTTCTCTATACATATTCAA GTTGGTTACC chA D130P-F, SEQ ID NO: 239GTAACCAACTTGAATCCATAT AGAGAAAAC chA D130P-R, SEQ ID NO: 240CGTTTTCTCTATATGGATTCAA GTTGGTTACC chA D130Q-F, SEQ ID NO: 241GTAACCAACTTGAATCAATAT AGAGAAAAC chA D130Q-R, SEQ ID NO: 242CGTTTTCTCTATATTGATTCAA GTTGGTTACC chA D130R-F, SEQ ID NO: 243GTAACCAACTTGAATAGATAT AGAGAAAAC chA D130R-R, SEQ ID NO: 244CGTTTTCTCTATATCTATTCAA GTTGGTTACC chA D130S-F, SEQ ID NO: 245GTAACCAACTTGAATAGCTAT AGAGAAAAC chA D130S-R, SEQ ID NO: 246CGTTTTCTCTATAGCTATTCAA GTTGGTTACC chA D130T-F, SEQ ID NO: 247GTAACCAACTTGAATACATAT AGAGAAAAC chA D130T-R, SEQ ID NO: 248CGTTTTCTCTATATGTATTCAA GTTGGTTACC chA D130V-F, SEQ ID NO: 249GTAACCAACTTGAATGTATAT AGAGAAAAC chA D130V-R, SEQ ID NO: 250CGTTTTCTCTATATACATTCAA GTTGGTTACC chA D130W-F, SEQ ID NO: 251GTAACCAACTTGAATTGGTAT AGAGAAAAC chA D130W-R, SEQ ID NO: 252CGTTTTCTCTATACCAATTCAA GTTGGTTACC chA D130Y-F, SEQ ID NO: 253GTAACCAACTTGAATTACTAT AGAGAAAAC chA D130Y-R, SEQ ID NO: 254CGTTTTCTCTATAGTAATTCAA GTTGGTTACC chA D130I-F, SEQ ID NO: 255GTAACCAACTTGAATATCTAT AGAGAAAAC chA D130I-R, SEQ ID NO: 256CGTTTTCTCTATAGATATTCAA GTTGGTTACC

Example 15: Influence of the 130-Site Mutant of chANP32A Protein on theReplication of Influenza Virus H7N9_(ZJ13)

DKO cells constructed in Example 2 were plated in a 12-well plate at3×10⁵/well; after 20 hours, the 130-site mutant of chANP32A constructedin Example 14 were co-transfected with the 6 plasmids of H7N9_(ZJ13)polymerase reporter system. The transfection system was: PB1 (80 ng),PB2 (80 ng), PA (40 ng), NP (160 ng), pMD 18T-vLuc (80 ng), pRL-TK (10ng) and the plasmid of ANP32 mutant protein (20 ng); and the emptyvector (20 ng) was set as a negative control, chANP32A (20 ng) was setas positive control, and each group was provided with triplicatewells.24 hours after transfection, the cells were lysed and the activityof polymerase was detected; The result showed that: compared withchANP32A, the two-point mutant of chANP32A N129I/D130N and thesingle-point mutants of chANP32A D130V, chANP32A D130F, chANP32A D130W,chANP32A D130H, chANP32A D130R, chANP32A D130K and chANP32A D130Y didnot have the ability to support H7N9_(ZJ13) polymerase activity; whilechANP32A D130A, chANP32A D130G, chANP32A D130C and chANP32A D130E allsupported H7N9_(ZJ13) polymerase activity; the ability of chANP32A D130Sand chANP32A D130T to support polymerase activity was reduced byapproximately 3 times; the ability of chANP32A D130L, chANP32A D130P,chANP32A D1301, chANP32A D130M, chANP32A D130Q and chANP32A D130N tosupport H7N9_(ZJ13) polymerase activity was reduced by approximately10-50 times; the result was shown in FIG. 25.

Example 16: Influence of the 130-Site Mutant of chANP32A Protein on theReplication of Influenza Virus H7N9_(AH13)

DKO cells constructed in Example 2 were plated in a 12-well plate at3×10⁵/well; after 20 hours, the 130-site mutant of chANP32A constructedin Example 14 were co-transfected with the 6 plasmids of H7N9_(AH13)polymerase reporter system. The transfection system was: PB1 (80 ng),PB2 (80 ng), PA (40 ng), NP (160 ng), pMD18T-vLuc (80 ng), pRL-TK (10ng) and the plasmid of ANP32 mutant protein (20 ng); and the emptyvector (20 ng) was set as a negative control, chANP32A (20 ng) was setas positive control, and each group was provided with triplicatewells.24 hours after transfection, the cells were lysed and the activityof polymerase was detected; The result showed that: compared withchANP32A, the two-point mutant of chANP32A N129I/D130N and thesingle-point mutants of chANP32A D130F and chANP32A D130K did not havethe ability to support H7N9_(AH13) polymerase activity; while chANP32AD130A, chANP32A D130S, chANP32A D130G and chANP32A D130E all supportedH7N9_(AH13) polymerase activity; the ability of chANP32A D130V andchANP32A D130R to support polymerase activity was reduced by more than100 times, and almost did not have the ability to support polymeraseactivity; the ability of chANP32A D130L, chANP32A D130P, chANP32A D1301,chANP32A D130M, chANP32A D130W, chANP32A D130H, chANP32A D130Q andchANP32A D130Y to support H7N9_(AH13) polymerase activity was reduced byapproximately 10-100 times; the ability of chANP32A D130T, chANP32AD130C and chANP32A D130N to support H7N9_(AH13) polymerase activity wasreduced by approximately 3-5 times; the result was shown in FIG. 26.

Example 17: Influence of the 130-Site Mutant of chANP32A Protein on theReplication of Influenza Virus WSN

DKO cells constructed in Example 2 were plated in a 12-well plate at3×10⁵/well; after 20 hours, the 130-site mutant of chANP32A constructedin Example 14 was co-transfected with the 6 plasmids of WSN polymerasereporter system. The transfection system was: PB1 (80 ng), PB2 (80 ng),PA (40 ng), NP (160 ng), pMD18T-vLuc (80 ng), pRL-TK (10 ng) and theplasmid of ANP32 mutant protein (20 ng); and the empty vector (20 ng)was set as a negative control, chANP32A (20 ng) was set as positivecontrol, and each group was provided with triplicate wells.24 hoursafter transfection, the cells were lysed and the activity of polymerasewas detected; The result showed that: compared with chANP32A, thetwo-point mutant of chANP32A N129I/D130N and the single-point mutants ofchANP32A D130F, chANP32A D130R and chANP32A D130K did not have theability to support WSN polymerase activity; while chANP32A D130S andchANP32A D130G, chANP32A D130E all supported WSN polymerase activity;chANP32A D130V, chANP32A D130W, chANP32A D130H and chANP32A D130Y almostdid not have the ability to support polymerase activity; the ability ofchANP32A D130L, chANP32A D130P, chANP32A D1301, chANP32A D130M, chANP32AD130Q and chANP32A D130N to support WSN polymerase activity was reducedby approximately 10-50 times; the ability of chANP32A D130A, chANP32AD130T and chANP32A D130C to support WSN polymerase activity was reducedby approximately 2-3 times; the result was shown in FIG. 27.

Example 18: Construction of the Vector of huANP32B Protein SegmentedMutation and Determination of Polymerase Activity

TABLE 21 primer sequences of huANP32B protein segmented mutationprimer name sequence (5′-3′) huANP_B1_F, TCGCGGCCGCATGGCCGCCGCCGCCGCCGCCSEQ ID NO: GCCGCCGCCCTGAGGAACCGGACCCCG 257 Human_B1_R,GGTTCCTCAGGGCGGCGGCGGCGGCGGCGG SEQ ID NO: CGGCGGCCATGCGGCCGCGAGCTCGAA258 Human_B2_F, CCACCTGGAGGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCGAACTTGTCTTGGACAAT 259 Human_B2_R,AGACAAGTTCGGCGGCGGCGGCGGCGGCGG SEQ ID NO: CGGCGGCGGCCTCCAGGTGGATCCTCCT260 Human_B3_F, AGCTGTTCGAGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCGATGGAAAAATTGAGGGC 261 Human_B3_R,TTTTTCCATCGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCTCGAACAGCTGCCGGGGT262 Human_B4_F, CAAATCAAATGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCTTTGTGAACTTAGAGTTC 263 Human_B4_R,AGTTCACAAAGGCGGCGGCGGCGGCGGCGG SEQ ID NO: CGGCGGCGGCATTTGATTTGCAATTGTC264 Human_B5_F, AACAGCTGAAGCCGCCGCCGCCGCCGCCGC SEQ ID NO:CGCCGCCGCCAATGTAGGCTTGATCTCA 265 Human_B5_R,AGCCTACATTGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCTTCAGCTGTTAAGCCCTC266 Human_B6_F, CAGTTTAATAGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCCCCAAGCTGCCTAAATTG 267 Human_B6_R,GCAGCTTGGGGGCGGCGGCGGCGGCGGCGG SEQ ID NO: CGGCGGCGGCTATTAAACTGAGGAACTC268 Human_B7_F, TTCAAATCTCGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCCTCAGTGAAAATAGAATC 269 Human_B7_R,TTTCACTGAGGGCGGCGGCGGCGGCGGCGG SEQ ID NO: CGGCGGCGGCGAGATTTGAAACTGAGAT270 Human_B8_F, AAAGCTTGAAGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCGACATGTTAGCTGAAAAA 271 Human_B8_R,CTAACATGTCGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCTTCAAGCTTTTTCAATTT272 Human_B9_F, TGGAGGTCTGGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCACACATCTAAACTTAAGT 273 Human_B9_R,TTAGATGTGTGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCCAGACCTCCAAAGATTCT274 Human_B10_F, TCCAAATCTCGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCAAAGATATCAGCACCTTG 275 Human_B10_R,TGATATCTTTGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCGAGATTTGGAAGTTTTTC276 Human_B11_F, AAATAAACTGGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCAAGTTAGAATGTCTGAAA 277 Human_B11_R,ATTCTAACTTGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCCAGTTTATTTCCACTTAA278 Human_B12_F, ACCTTTGAAAGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCTTTAACTGTGAGGTTACC 279 Human_B12_R,CACAGTTAAAGGCGGCGGCGGCGGCGGCGG SEQ ID NO: CGGCGGCGGCTTTCAAAGGTTCCAAGGT280 Human_B13_F, CCTGGACCTCGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCTACCGAGAGAGTGTCTTC 281 Human_B13_R,TCTCTCGGTAGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCGAGGTCCAGGCTTTTCAG282 Human_B14_F, CCTGAATGACGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCCAGCTTACCTACTTGGAT 283 Human_B14_R,AGGTAAGCTGGGCGGCGGCGGCGGCGGCGG SEQ ID NO: CGGCGGCGGCGTCATTCAGGTTGGTAAC284 Human_B15_F, GCTCCTGCCCGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCGAGGACCAGGAAGCACCT 285 Human_B15_R,CCTGGTCCTCGGCGGCGGCGGCGGCGGCGG SEQ ID NO: CGGCGGCGGCGGGCAGGAGCTTGAAGAC286 Human_B16_F, CTATGACCGAGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCGAGGTGGATGGTGTGGAT 287 Human_B16_R,CATCCACCTCGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCTCGGTCATAGCCATCCAA288 Human_B17_F, CTCAGATGCCGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCGACGAAGAAGGAGAAGAT 289 Human_B17_R,CTTCTTCGTCGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCGGCATCTGAGTCAGGTGC290 Human_B18_F, AGAGGAGGAGGCCGCCGCCGCCGCCGCCGC SEQ ID NO:CGCCGCCGCCGACGATGAGGATGGTGAA 291 Human_B18_R,CCTCATCGTCGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCCTCCTCCTCTTCATCCAC292 Human_B19_F, GGAAGACGAGGCCGCCGCCGCCGCCGCCGC SEQ ID NO:CGCCGCCGCCGATGAAGAAGATGATGAA 293 Human_B19_R,CTTCTTCATCGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCCTCGTCTTCCTCATCTTC294 Human_B20_F, AGAGGAGTTTGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCGAAGGGGATGAGGACGAC 295 Human_B20_R,CATCCCCTTCGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCAAACTCCTCTTCTTCACC296 Human_B21_F, TGAAGATGTAGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCGAGGAGGAAGAAGAATTT 297 Human_B21_R,CTTCCTCCTCGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCTACATCTTCATCTTCATC298 Human_B22_F, TGAAGTCAGTGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCGAAGATGAAGATGAGGAT 299 Human_B22_R,CTTCATCTTCGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCACTGACTTCATCGTCGTC300 Human_B23_F, ACTTGATGAAGCCGCCGCCGCCGCCGCCGCC SEQ ID NO:GCCGCCGCCGAGGAAGAAGGTGGGAAA 301 Human_B23_R,CTTCTTCCTCGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCTTCATCAAGTCCAAATTC302 Human_B24_F, GGATGAAGAGGCCGCCGCCGCCGCCGCCGC SEQ ID NO:CGCCGCCGCCAAGAGAGAAACAGATGA 303 Human_B24_R,TTTCTCTCTTGGCGGCGGCGGCGGCGGCGGC SEQ ID NO: GGCGGCGGCCTCTTCATCCTCATCCTC304 Human_B25_F, TGAAAAGAGGGCCGCCGCCGCCGCCGCCGC SEQ ID NO:CGCCGCCGCCGCCGGCAGCGGAGACTACA 305 Human_B25_R,CTCCGCTGCCGGCGGCGGCGGCGGCGGCGG SEQ ID NO: CGGCGGCGGCGGCCCTCTTTTCACCTTT306

The huANP32B protein was subjected to a segmented mutation, whereinevery 10 amino acids as a group were uniformly mutated to alanine, andthe primers for point mutation were shown in Table 21 (the mutated baseswere underlined). For example, the huANP32B B1-10A mutant was resultedfrom the mutation of amino acid segment DMKRRIHLE at positions 2-9 ofhuANP32B protein to AAAAAAAAA, and the huANP32B B11-20A mutant wasresulted from the mutation of amino acid segment LRNRTPAAVR at positions11-20 of huANP32B protein to AAAAAAAAAA, and so on. Using KOD-FXhigh-efficiency DNA polymerase and using PCAGGS-huANP32B as a template,the following segmented mutants were respectively constructed: huANP32BB1-10A (using primer pair SEQ ID NO: 257 and SEQ ID NO: 258), huANP32BB11-20A (using primer pair SEQ ID NO: 259 and SEQ ID NO: 260), huANP32BB21-30A (using primer pair SEQ ID NO: 261 and SEQ ID NO: 262), huANP32BB31-40A (using primer pair SEQ ID NO: 263 and SEQ ID NO: 264), huANP32BB41-50A (using primer pair SEQ ID NO: 265 and SEQ ID NO: 266), huANP32BB51-60A (using primer pair SEQ ID NO: 267 and SEQ ID NO: 268), huANP32BB61-70A (using primer pair SEQ ID NO: 269 and SEQ ID NO: 270), huANP32BB71-80A (using primer pair SEQ ID NO: 271 and SEQ ID NO: 272), huANP32BB81-90A (using primer pair SEQ ID NO: 273 and SEQ ID NO: 274), huANP32BB91-100A (using primer pair SEQ ID NO: 275 and SEQ ID NO: 276), huANP32BB101-110A (using primer pair SEQ ID NO: 277 and SEQ ID NO: 278),huANP32B B111-120A (using primer pair SEQ ID NO: 279 and SEQ ID NO:280), huANP32B B121-130A (using primer pair SEQ ID NO: 281 and SEQ IDNO: 282), huANP32B B131-140A (using primer pair SEQ ID NO: 283 and SEQID NO: 284), huANP32B B141-150A (using primer pair SEQ ID NO: 285 andSEQ ID NO: 286), huANP32B B151-160tA (using primer pair SEQ ID NO: 287and SEQ ID NO: 288), huANP32B B161-170A (using primer pair SEQ ID NO:289 and SEQ ID NO: 290), huANP32B B171-180A (using primer pair SEQ IDNO: 291 and SEQ ID NO: 292), huANP32B B181-190A (using primer pairs SEQID NO: 293 and SEQ ID NO: 294), huANP32B B191-200A (using primer pairSEQ ID NO: 295 and SEQ ID NO: 296), huANP32B B201-210A (using primerpairs SEQ ID NO: 297 and SEQ ID NO: 298), huANP32B B211-220A (usingprimer pair SEQ ID NO: 299 and SEQ ID NO: 300), huANP32B B221-230A(using primer pair SEQ ID NO: 301 and SEQ ID NO: 302), huANP32BB231-240A (using primer pair SEQ ID NO: 303 and SEQ ID NO: 304),huANP32B B241-251A (using primer pair SEQ ID NO: 305 and SEQ ID NO:306), and were respectively named as huANP32B B1-10A, huANP32B B11-20A,huANP32B B21-30A, huANP32B B31-40A, huANP32B B41-50A, huANP32B B51-60A,huANP32B B61-70A, huANP32B B71-80A, huANP32B B81-90A, huANP32B B91-100A,huANP32B B101-110A, huANP32B B111-120A, huANP32B B121-130A, huANP32BB131-140A, huANP32B B141-150A, huANP32B B151-160A, huANP32B B161-170A,huANP32B B171-180A, huANP32B B181-190A, huANP32B B191-200A, huANP32BB201-210A, huANP32B B211-220A, huANP32B B221-230A, huANP32B B231-240Aand huANP32B B241-251A. The obtained PCR product was digested with Dpn Iin a 37° C. constant temperature water bath for 30 minutes, and then 2.5ul of the digested product was taken and transformed into 20 ul of DH5αcompetent cells; the next day, a single clone was picked for sequencing,and the plasmid which was verified correct by sequencing was used forsubsequent transfection experiment.

Example 19: Influence of the Segmented Mutant of huANP32B Protein on theReplication of Influenza Virus H7N9_(AH13)

Double-knockout cell lines (DKO) were plated in a 24-well plate at1×10⁵/well; after 20 hours, the segmented mutant of huANP32B constructedin Example 18 were co-transfected with the 6 plasmids of H7N9_(AH13)polymerase reporter system. The transfection system was: PB1 (40 ng),PB2 (40 ng), PA (20 ng), NP (80 ng), pMD18T-vLuc (40 ng), pRL-TK (5 ng)and the plasmid of ANP32 mutant protein (10 ng); and the empty vector(10 ng) was set as a negative control, huANP32B (10 ng) was set aspositive control, and each group was provided with triplicate wells.24hours after transfection, the cells were lysed and the activity ofpolymerase was detected; the result showed that: compared with huANP32B,the segmented mutants of huANP32B B51-60A, huANP32B B61-70A, huANP32BB71-80A, huANP32B B81-90A, huANP32B B91-100A, huANP32B B101-110A,huANP32B B111-120A, huANP32B B121-130A, huANP32B B131-140A, huANP32BB141-150A and huANP32B B151-160A did not have the ability to supportH7N9_(AH13) polymerase activity; while huANP32B B1-10A, huANP32BB11-20A, huANP32B B21-30A, huANP32B B31-40A, huANP32B B171-180A,huANP32B B181-190A, huANP32B B191-200A, huANP32B B201-210A, huANP32BB211-220A, huANP32B B221-230A, huANP32B B231-240A and huANP32B B241-251Aall had the ability to support H7N9_(AH13) polymerase activity; theability of huANP32B B41-50A and huANP32B B161-170A to supportH7N9_(AH13) polymerase activity was reduced by about 200 times; theresult was shown in FIG. 28.

Example 20: Construction of the Vector of chANP32A Protein SegmentedMutation and Determination of Polymerase Activity

TABLE 22 primer sequences used for chANP32A protein segmented mutationname sequence (5′-3′) CK32A_B1_F, SEQ ID TCGCGGCCGCATGGCCGCCGCCGCCGNO: 307 CCGCCGCCGCCGCCCTGCGGAACAGG ACGCCCT CK32A_B1_R, SEQ IDTCCTGTTCCGCAGGGCGGCGGCGGCG NO: 308 GCGGCGGCGGCGGCCATGCGGCCGCG AGCTCGAACK32A_B2_F, SEQ ID GGATCCACTTAGAGGCCGCCGCCGCC NO: 309GCCGCCGCCGCCGCCGCCGAACTTGTT CTTGAC CK32A_B2_R, SEQ IDAAGAACAAGTTCGGCGGCGGCGGCGG NO: 310 CGGCGGCGGCGGCGGCCTCTAAGTGG ATCCTTCK32A_B3_F, SEQ ID CAGATGTTAAGGCCGCCGCCGCCGCC NO: 311GCCGCCGCCGCCGCCGAAGGCAAAAT TGAAGG CK32A_B3_R, SEQ IDAATTTTGCCTTCGGCGGCGGCGGCGGC NO: 312 GGCGGCGGCGGCGGCCTTAACATCTG AGGGCCK32A_B4_F, SEQ ID CTGTAGGTCATACGCCGCCGCCGCCGC NO: 313CGCCGCCGCCGCCGCCTTTGAAGAGC TGGAAT CK32A_B4_R, SEQ IDAGCTCTTCAAAGGCGGCGGCGGCGGC NO: 314 GGCGGCGGCGGCGGCGTATGACCTAC AGTTGTCK32A_B5_F, SEQ ID TTACAGATGAGGCCGCCGCCGCCGCC NO: 315GCCGCCGCCGCCGCCAACGTAGGCTTA GCCTC CK32A_B5_R, SEQ IDTAAGCCTACGTTGGCGGCGGCGGCGG NO: 316 CGGCGGCGGCGGCGGCCTCATCTGTA AGGCCTCK32A_B6_F, SEQ ID TGAGTACAATCGCCGCCGCCGCCGCCG NO: 317CCGCCGCCGCCGCCCCAAAGTTAAAC AAACT CK32A_B6_R, SEQ IDTTAACTTTGGGGCGGCGGCGGCGGCG NO: 318 GCGGCGGCGGCGGCGATTGTACTCAA GAATTCCCK32A_B7_F, SEQ ID TGCAAACTTAGCCGCCGCCGCCGCCG NO: 319CCGCCGCCGCCGCCCTAAGTGACAAC AGAGTC CK32A_B7_R, SEQ IDTTGTCACTTAGGGCGGCGGCGGCGGC NO: 320 GGCGGCGGCGGCGGCTAAGTTTGCAA CTGAGGCK32A_B8_F, SEQ ID GAAGCTCGAAGCCGCCGCCGCCGCCG NO: 321CCGCCGCCGCCGCCGAAGTGTTGGCA GAAAAG CK32A_B8_R, SEQ IDCCAACACTTCGGCGGCGGCGGCGGCG NO: 322 GCGGCGGCGGCGGCTTCGAGCTTCTTA AGTTTCK32A_B9_F, SEQ ID AGGAGGACTGGCCGCCGCCGCCGCCG NO: 323CCGCCGCCGCCGCCACGCATCTAAATC TAAGT CK32A_B9_R, SEQ IDTTTAGATGCGTGGCGGCGGCGGCGGC NO: 324 GGCGGCGGCGGCGGCCAGTCCTCCTG AGACTCK32A_B10_F, SEQ TCCAAACCTCGCCGCCGCCGCCGCCG ID NO: 325CCGCCGCCGCCGCCAAAGATCTTGGTA CAATA CK32A_B10_R, SEQCCAAGATCTTTGGCGGCGGCGGCGGC ID NO: 326 GGCGGCGGCGGCGGCGAGGTTTGGAC ACTTTTCK32A_B11_F, SEQ GGCAACAAAATAGCCGCCGCCGCCGC ID NO: 327CGCCGCCGCCGCCGCCAAGTTAGAAA ACCTGA CK32A_B11_R, SEQTTTTCTAACTTGGCGGCGGCGGCGGCG ID NO: 328 GCGGCGGCGGCGGCTATTTTGTTGCCA CTTACK32A_B12_F, SEQ ACCTCTGAAAGCCGCCGCCGCCGCCG ID NO: 329CCGCCGCCGCCGCCTTCAATTGCGAGG TAACC CK32A_B12_R, SEQCGCAATTGAAGGCGGCGGCGGCGGCG ID NO: 330 GCGGCGGCGGCGGCTTTCAGAGGTTC TATTGTCK32A_B13_F, SEQ TTTAGATCTTGCCGCCGCCGCCGCCGC ID NO: 331CGCCGCCGCCGCCTATAGAGAAAACGT ATTC CK32A_B13_R, SEQTTTCTCTATAGGCGGCGGCGGCGGCGG ID NO: 332 CGGCGGCGGCGGCAAGATCTAAACTC TTCAGCK32A_B14_F, SEQ ACTTGAATGATGCCGCCGCCGCCGCCG ID NO: 333CCGCCGCCGCCGCCCAACTCACATACC TCGA CK32A_B14_R, SEQATGTGAGTTGGGCGGCGGCGGCGGCG ID NO: 334 GCGGCGGCGGCGGCATCATTCAAGTTG GTTACCK32A_B15_F, SEQ GCTCCTCCCAGCCGCCGCCGCCGCCGC ID NO: 335CGCCGCCGCCGCCGATGACAAAGAAG CACCA CK32A_B15_R, SEQTCTTTGTCATCGGCGGCGGCGGCGGCG ID NO: 336 GCGGCGGCGGCGGCTGGGAGGAGCTT GAATACK32A_B16_F, SEQ CTACGATCGGGCCGCCGCCGCCGCCGC ID NO: 337CGCCGCCGCCGCCGAGGGCTACGTGG AGGGC CK32A_B16_R, SEQCGTAGCCCTCGGCGGCGGCGGCGGCG ID NO: 338 GCGGCGGCGGCGGCCCGATCGTAGCC ATCGAGCK32A_B17_F, SEQ CTCTGATGCAGCCGCCGCCGCCGCCGC ID NO: 339CGCCGCCGCCGCCGAGGAAGATGAAG ATGTC CK32A_B17_R, SEQCATCTTCCTCGGCGGCGGCGGCGGCG ID NO: 340 GCGGCGGCGGCGGCTGCATCAGAGTC TGGTGCCK32A_B18_F, SEQ AGACGATGAGGCCGCCGCCGCCGCCG ID NO: 341CCGCCGCCGCCGCCAAAGATCGGGAT GACAAA CK32A_B18_R, SEQCCCGATCTTTGGCGGCGGCGGCGGCG ID NO: 342 GCGGCGGCGGCGGCCTCATCGTCTAA GCCCTCCK32A_B19_F, SEQ ATCTCTAGTGGCCGCCGCCGCCGCCGC ID NO: 343CGCCGCCGCCGCCTCTGATGCAGAGG GCTAC CK32A_B19_R, SEQCTGCATCAGAGGCGGCGGCGGCGGCG ID NO: 344 GCGGCGGCGGCGGCCACTAGAGATAA GACATCCK32A_B20_F, SEQ AGCACCGGACGCCGCCGCCGCCGCCG ID NO: 345CCGCCGCCGCCGCCGACGACGAGGAG GAAGAT CK32A_B20_R, SEQCCTCGTCGTCGGCGGCGGCGGCGGCG ID NO: 346 GCGGCGGCGGCGGCGTCCGGTGCTTC TTTGTCCK32A_B21_F, SEQ GGAAGGCTTAGCCGCCGCCGCCGCCG ID NO: 347CCGCCGCCGCCGCCGAGTATGACGATG ATGCT CK32A_B21_R, SEQCGTCATACTCGGCGGCGGCGGCGGCG ID NO: 348 GCGGCGGCGGCGGCTAAGCCTTCCAC GTAGCCCK32A_B22_F, SEQ AGACGAAGAGGCCGCCGCCGCCGCCG ID NO: 349CCGCCGCCGCCGCCGATGAAGAGGAT GAGGAG CK32A_B22_R, SEQCCTCTTCATCGGCGGCGGCGGCGGCG ID NO: 350 GCGGCGGCGGCGGCCTCTTCGTCTTCA TCTTCCK32A_B23_F, SEQ GGTAGTAGAAGCCGCCGCCGCCGCCG ID NO: 351CCGCCGCCGCCGCCGGAGAAGAGGAG GACGTA CK32A_B23_R, SEQCCTCTTCTCCGGCGGCGGCGGCGGCG ID NO: 352 GCGGCGGCGGCGGCTTCTACTACCTGA GCATCCK32A_B24_F, SEQ GGAAGAGGAAGCCGCCGCCGCCGCCG ID NO: 353CCGCCGCCGCCGCCGAGGAGGATGAG GAAGGC CK32A_B24_R, SEQCATCCTCCTCGGCGGCGGCGGCGGCG ID NO: 354 GCGGCGGCGGCGGCTTCCTCTTCCTCC TCCTCCK32A_B25_F, SEQ CGGAGAGGAAGCCGCCGCCGCCGCCG ID NO: 355CCGCCGCCGCCGCCGACGTAGATGATG ATGAA CK32A_B25_R, SEQCATCTACGTCGGCGGCGGCGGCGGCG ID NO: 356 GCGGCGGCGGCGGCTTCCTCTCCGCTT ACGTCCK32A_B26_F, SEQ TAATGATGGTGCCGCCGCCGCCGCCGC ID NO: 357CGCCGCCGCCGCCCCCGATGAAGAAC GGGGA CK32A_B26_R, SEQCTTCATCGGGGGCGGCGGCGGCGGCG ID NO: 358 GCGGCGGCGGCGGCACCATCATTATAG CCTTCCK32A_B27_F, SEQ TGAAGAAGAAGCCGCCGCCGCCGCCG ID NO: 359CCGCCGCCGCCGCCCGAGAACCCGAA GACGAA CK32A_B27_R, SEQCGGGTTCTCGGGCGGCGGCGGCGGCG ID NO: 360 GCGGCGGCGGCGGCTTCTTCTTCATCT TCATCCK32A_B28_F, SEQ GAAGAGGAAAGCCGCCGCCGCCGCCG ID NO: 361CCGCCGCCGCCGCCGCCGGCAGCGGA GACTAC CK32A_B28_R, SEQCTCCGCTGCCGGCGGCGGCGGCGGCG ID NO: 362 GCGGCGGCGGCGGCGGCTTTCCTCTTC TGTCC

The chANP32A protein was subjected to a segmented mutation, whereinevery 10 amino acids as a group were uniformly mutated to alanine, andthe primers for point mutation were shown in Table 22 (the mutated baseswere underlined). For example, the chANP32A 1-10A mutant was resultedfrom the mutation of amino acid segment DMKKRIHLE at positions 2-9 ofchANP32A protein to AAAAAAAAA, and the chANP32A 11-20A mutant is themutation of amino acid segment LRNRTPSDVK at positions 11-20 of chANP32Aprotein to AAAAAAAAAA, and so on. Using KOD-FX high-efficiency DNApolymerase and using PCAGGS-chANP32A as a template, the followingsegmented mutants were respectively constructed: chANP32A 1-10 mutA(using primer pair SEQ ID NO: 307 and SEQ ID NO: 308), chANP32A 11-20mutA (using primer pair SEQ ID NO: 309 and SEQ ID NO: 310), chANP32A21-30mutA (using primer pair SEQ ID NO: 311 and SEQ ID NO: 312),chANP32A 31-40mutA (using primer pair SEQ ID NO: 313 and SEQ ID NO:314), chANP32A 41-50mutA (using primer pair SEQ ID NO: 315 and SEQ IDNO: 316), chANP32A 51-60mutA (using primer pair SEQ ID NO: 317 and SEQID NO: 318), chANP32A 61-70mutA (using primer pair SEQ ID NO: 319 andSEQ ID NO: 320), chANP32A 71-80mutA (using primer pair SEQ ID NO: 321and SEQ ID NO: 322), chANP32A 81-90mutA (using primer pair SEQ ID NO:323 and SEQ ID NO: 324), chANP32A 91-100mutA (using primer pair SEQ IDNO: 325 and SEQ ID NO: 326), chANP32A 101-110mutA (using primer pair SEQID NO: 327 and SEQ ID NO: 328), chANP32A 111-120mutA (using primer pairSEQ ID NO: 329 and SEQ ID NO: 330), chANP32A 121-130mutA (using primerpair SEQ ID NO: 331 and SEQ ID NO: 332), chANP32A 131-140mutA (usingprimer pair SEQ ID NO: 333 and SEQ ID NO: 334), chANP32A 141-150mutA(using primer pair SEQ ID NO: 335 and SEQ ID NO: 336), chANP32A151-160mutA (using primer pairs SEQ ID NO: 337 and SEQ ID NO: 338),chANP32A 161-170mutA (using primer pair SEQ ID NO: 339 and SEQ ID NO:340), chANP32A 171-180mutA (using primer pair SEQ ID NO: 341 and SEQ IDNO: 342), chANP32A 181-190mutA (using primer pairs SEQ ID NO: 343 andSEQ ID NO: 344), chANP32A 191-200mutA (using primer pairs SEQ ID NO: 345and SEQ ID NO: 346), chANP32A 201-210mutA (using primer pairs SEQ ID NO:347 and SEQ ID NO: 348), chANP32A 211-220mutA (using primer pairs SEQ IDNO: 349 and SEQ ID NO: 350), chANP32A 221-230mutA (using primer pairsSEQ ID NO: 351 and SEQ ID NO: 352), chANP32A 231-240mutA (using primerpair SEQ ID NO: 353 and SEQ ID NO: 354), chANP32A 241-250mutA (usingprimer pair SEQ ID NO: 355 and SEQ ID NO: 356), chANP32A 251-260mutA(using primer pair SEQ ID NO: 357 and SEQ ID NO: 358), chANP32A261-270mutA (using primer pair SEQ ID NO: 359 and SEQ ID NO: 360),chANP32A 271-281mutA (using primer pair SEQ ID NO: 361 and SEQ ID NO:362), and were respectively named as chANP32A 1-10mutA, chANP32A11-20mutA, chANP32A 21-30mutA, chANP32A 31-40mutA, chANP32A 41-50mutA,chANP32A 51-60mutA, chANP32A 61-70mutA, chANP32A 71-80mutA, chANP32A81-90mutA, chANP32A 91-100mutA, chANP32A 101-110mutA, chANP32A111-120mutA, chANP32A 121-130mutA, chANP32A 131-140mutA, chANP32A141-150mutA, chANP32A 151-160mutA, chANP32A 161-170mutA, chANP32A171-180mutA, chANP32A 181-190mutA, chANP32A 191-200mutA, chANP32A201-210mutA, chANP32A 211-220mutA, chANP32A 221-230mutA, chANP32A231-240mutA, chANP32A 241-250mutA, chANP32A 251-260mutA, chANP32A261-270mutA and chANP32A 271-281mutA. The obtained PCR product wasdigested with Dpn I in a 37° C. constant temperature water bath for 30minutes, and then 2.5 ul of the digested product was taken andtransformed into 20 ul of DH5α competent cells; the next day, a singleclone was picked for sequencing, and the plasmid which was verifiedcorrect by sequencing was used for subsequent transfection experiment.

Example 21: Influence of the Segmented Mutant of chANP32A Protein on theReplication of Influenza Virus H7N9_(ZJ13)

Influence of the segmented mutant of chANP32A protein on the replicationof influenza virus H7N9_(ZJ13): double-knockout cell lines (DKO) wereplated in a 24-well plate at 1×10⁵/well; after 20 hours, the segmentedmutant of chANP32A constructed in Example 20 were co-transfected withthe 6 plasmids of H7N9_(ZJ13) polymerase reporter system. Thetransfection system was: PB1 (40 ng), PB2 (40 ng), PA (20 ng), NP (80ng), pMD18T-vLuc (40 ng), pRL-TK (5 ng) and the plasmid of ANP32 mutantprotein (10 ng); and the empty vector (10 ng) was set as a negativecontrol, chANP32A (10 ng) was set as positive control, and each groupwas provided with triplicate wells.24 hours after transfection, thecells were lysed and the activity of polymerase was detected, and theresults showed that: compared with chANP32A, the segmented mutantschANP32A 71-80mutA, chANP32A 81-90mutA, chANP32A 91-100mutA, chANP32A101-110mutA, chANP32A 111-120mutA, chANP32A 121-130mutA, chANP32A131-140mutA, chANP32A 141-150mutA, chANP32A 151-160mutA, chANP32A161-170mutA and chANP32A 171-180mutA did not have the ability to supportH7N9_(ZJ13) polymerase activity; while chANP32A 1-10mutA,chANP32A11-20mutA, chANP32A 21-30mutA, chANP32A 31-40mutA, chANP32A41-50mutA, chANP32A 51-60mutA, chANP32A 181-190mutA, chANP32A201-210mutA, chANP32A 211-220mutA, chANP32A 221-230mutA, chANP32A231-240mutA, chANP32A 241-250mutA, chANP32A 251-260mutA, chANP32A261-270mutA, chANP32A 271-281mutA all supported H7N9_(ZJ13) polymeraseactivity; the ability of chANP32A 61-70mutA and chANP32A 191-200mutA tosupport H7N9_(ZJ13) polymerase activity was reduced by about 100 times;the result was shown in FIG. 29.

Example 22: Construction of the Amino Acid Site Mutation Vector ofchANP32A Protein and Determination of Polymerase Activity

TABLE 23 primer sequences of chANP32A protein amino acid site mutationname sequence (5′-3′) chA_D149A_F, SEQ ID TTATCTCTAGTGAAAGCCCGGGATGANO: 363 CAAAGAA chA_D149A_R, SEQ ID TTCTTTGTCATCCCGGGCTTTCACTAG NO: 364AGATAA chA_R150A_F, SEQ ID TCTCTAGTGAAAGATGCCGATGACAA NO: 365 AGAAGCAchA_R150A_R, SEQ ID TGCTTCTTTGTCATCGGCATCTTTCAC NO: 366 TAGAGAchA_D151A_F, SEQ ID CTAGTGAAAGATCGGGCCGACAAAG NO: 367 AAGCACCGchA_D151A_R, SEQ ID CGGTGCTTCTTTGTCGGCCCGATCTTT NO: 368 CACTAGchA_D152A_F, SEQ ID GTGAAAGATCGGGATGCCAAAGAAG NO: 369 CACCGGACchA_D152A_R, SEQ ID GTCCGGTGCTTCTTTGGCATCCCGAT NO: 370 CTTTCACchA_K153A_F, SEQ ID AAAGATCGGGATGACGCCGAAGCAC NO: 371 CGGACTCTchA_K153A_R, SEQ ID AGAGTCCGGTGCTTCGGCGTCATCCC NO: 372 GATCTTTchA_E154A_F, SEQ ID GATCGGGATGACAAAGCCGCACCGG NO: 373 ACTCTGATchA_E154A_R, SEQ ID ATCAGAGTCCGGTGCGGCTTTGTCAT NO: 374 CCCGATC

The primers of point mutation were shown in Table 23 (mutated bases wereunderlined);. using KOD-FX high-efficiency DNA polymerase and usingPCAGGS-chANP32A as a template, the following point mutants of chANP32Awere constructed: D149A (using primer pair SEQ ID NO: 363 and SEQ ID NO:364), R150A (using primer pair SEQ ID NO: 365 and SEQ ID NO: 366), D151A(using primer pair SEQ ID NO: 367 and SEQ ID NO: 368), D152A (usingprimer pair SEQ ID NO: 369 and SEQ ID NO: 370), K153A (using primer pairSEQ ID NO: 371 and SEQ ID NO: 372) and E154A (using primer pair SEQ IDNO: 373 and SEQ ID NO: 374), and were respectively named as chANP32AD149A, chANP32A R150A, chANP32A D151A, chANP32A D152A, chANP32A K153Aand chANP32A E154A. The obtained PCR product was digested with Dpn I ina 37° C. constant temperature water bath for 30 minutes, and then 2.5 ulof the digested product was taken and transformed into 20 ul of DH5αcompetent cells; the next day, a single clone was picked for sequencing,and the plasmid which was verified correct by sequencing was used forsubsequent transfection experiment.

Example 23: Influence of the chANP32A Protein Point Mutant on theReplication of Influenza Virus H7N9_(AH13)

Double-knockout cell lines (DKO) were plated in a 24-well plate at1×10⁵/well; after 20 hours, the amino acid mutant of chANP32Aconstructed in Example 22 were co-transfected with the 6 plasmids ofH7N9_(AH13) polymerase reporter system. The transfection system was: PB1(40 ng), PB2 (40 ng), PA (20 ng), NP (80 ng), pMD18T-vLuc (40 ng),pRL-TK (5 ng) and the plasmid of ANP32 mutant protein (10 ng); and theempty vector (10 ng) was set as a negative control, chANP32A (10 ng) wasset as positive control, and each group was provided with triplicatewells.24 hours after transfection, the cells were lysed and the activityof polymerase was detected; the result showed that: compared withchANP32A, the ability of chANP32A D149A to support H7N9_(AH13)polymerase activity was reduced by about 1000 times, and almost did nothave the ability to support the polymerase activity; the ability ofchANP32A D151A to support H7N9_(AH13) polymerase activity was reduced byabout 50 times; chANP32A R150A, chANP32A D152A and chANP32A K153A allsupported the H7N9_(AH13) polymerase activity; the ability of chANP32AE154A to support polymerase activity was reduced by about 5 times; theresult was shown in FIG. 30.

Example 24: Construction of the Amino Acid Site Mutation Vector ofhuANP32B Protein and Determination of Polymerase Activity

TABLE 24 primer sequences of huANP32B protein amino acid site mutationname sequence (5′-3′) huB_NES1_F, SEQ ID TGATCTCAGTTTCAAATGCCCCCAAGGNO: 375 CCCCTAAATTGAAAAAGCTTGAACTC AGTGA huB_NES1_R, SEQ IDAAGCTTTTTCAATTTAGGGGCCTTGGG NO: 376 GGCATTTGAAACTGAGATCAAGCCTAC ATTThuB_NES2_F, SEQ ID AGCTGAAAAAGCCCCAAATGCCACAC NO: 377ATGCCAACGCCAGTGGAAATAAACTG AAAGA huB_NES2_R, SEQ IDTTATTTCCACTGGCGTTGGCATGTGTG NO: 378 GCATTTGGGGCTTTTTCAGCTAACATG TCCAhuB_NES3_F, SEQ ID GAACCTTTGAAAAAGGCCGAATGTGC NO: 379CAAAAGCGCCGACCTCTTTAACTGTG AGGTT huB_NES3_R, SEQ IDTTAAAGAGGTCGGCGCTTTTGGCACAT NO: 380 TCGGCCTTTTTCAAAGGTTCCAAGGTG CTG

The protein sequence of huANP32B was analyzed and it contained 3 knownNuclear Export Signals (NES), which were NSE1(LPKLPKLKKL, located atpositions 60-71), NSE2 (LPNLTHLNL, located at positions 87-95) and NES3(LEPLKKLECLKSLDL, located at positions 106-120). To determine whetherthe nuclear export domain of huANP32B was correlated with its ability tosupport polymerase activity, mutations were made to these three nuclearexport regions, respectively. For the NES1 region, leucines at positions60 and 63 were both mutated to alanine, and the mutant was named ashuANP32B NES1mut. For the NES2 region, leucines at positions 87, 90, 93and 95 were all mutated to alanine, and the mutant was named as huANP32BNES2mut. For the NES3 region, leucines at positions 112, 115 and 118were all mutated to alanine, and the mutant was named as huANP32BNES3mut. The primers of point mutation were shown in Table 24 (mutatedbases were underlined);. using KOD-FX high-efficiency DNA polymerase foramplification and using PCAGGS-huANP32B as a template, the followingpoint mutants of huANP32B were constructed: NES1mut (using primer pairSEQ ID NO: 375 and SEQ ID NO: 376), NES2mut (using primer pair SEQ IDNO: 377 and SEQ ID NO: 378) and NES3mut (using primer pair SEQ ID NO:379 and SEQ ID NO: 380), and were respectively named as huANP32BNES1mut, huANP32B NES2mut and huANP32B NES3mut. The obtained PCR productwas digested with Dpn I in a 37° C. constant temperature water bath for30 minutes, and then 2.5 ul of the digested product was taken andtransformed into 20 ul of DH5α competent cells; the next day, a singleclone was picked for sequencing, and the plasmid which was verifiedcorrect by sequencing was used for subsequent transfection experiment.

Example 25: Influence of the Point Mutant of huANP32B Protein on theReplication of Influenza Virus H7N9_(AH13)

Double-knockout cell lines (DKO) were plated in a 24-well plate at1×10⁵/well; after 20 hours, the amino acid mutant of huANP32Bconstructed in Example 24 were co-transfected with the 6 plasmids ofH7N9_(AH13) polymerase reporter system. The transfection system was: PB1(40 ng), PB2 (40 ng), PA (20 ng), NP (80 ng), pMD18T-vLuc (40 ng),pRL-TK (5 ng) and the plasmid of ANP32 mutant protein (10 ng); and theempty vector (10 ng) was set as a negative control, huANP32B (10 ng) wasset as positive control, and each group was provided with triplicatewells.24 hours after transfection, the cells were lysed and the activityof polymerase was detected; the result showed that: compared withhuANP32B, the ability of huANP32B NES1mut to support H7N9_(AH13)polymerase activity was reduced by about 1000 times, and almost did nothave the ability to support the polymerase activity; huANP32B NES2mutand huANP32B NES3mut did not have the ability to support H7N9_(AH13)polymerase activity; the result was shown in FIG. 31.

Example 26: Construction of a Site-Directed Mutant Cell Line

We performed the construction of a site-directed mutant cell line byusing CRISPR-Cas9 technology. According to NCBI published referencenucleotide sequences human ANP32A (NM_006305.3) and human ANP32B(NM_006401.2), sgRNAs for positions 129/130 of the two proteins weredesigned by using the online software http://crispr.mit.edu/ (see Table25 for sequences).

TABLE 25 sgRNA sequences primer name primer sequence (5′-3′)huANP32A-129/130-sgRNA, CCAACCTGAACGACTACCGA SEQ ID NO: 381huANP32B-129/130-sgRNA, CTCTCGGTAGTCATTCAGGT SEQ ID NO: 382

sgRNA primers for the huANP32A 129/130 amino acid site and thehuANP32B129/130 amino acid site were designed; using the recombinantplasmid pMD18T-U6-huANPsgRNA-1 constructed in Example 2 as a templateand using KOD-FX Neo high-efficiency DNA polymerase (cat # KFX-201,purchased from Toyobo) for amplification, recombinant plasmidspMD18T-U6-huANP32A-129/130-sgRNA (using primer pair SEQ ID NO:383 andSEQ ID NO: 384) and pMD18T-U6-huANP32B-129/130-sgRNA (using primer pairSEQ ID NO:386 and SEQ ID NO: 387) were respectively constructed by apoint mutation PCR method, and the obtained PCR product was digestedwith Dpn I in a 37° C. constant temperature water bath for 30 minutes,then 5 ul of the digested product was taken and transformed into 20 ulof DH5α competent cells; the next day, a single clone was picked forsequencing, and the plasmids which were verified correct by sequencing,pMD18T-U6-huaNP32A-129/130-sgRNA (containing huANP 32A-129/130-sgRNA)and pMD18T-U6-huANP32B-129/130-sgRNA (containing huANP32B-129/130-sgRNA), were used for subsequent transfection experiment. Atthe same time, a donor sequence huANP32A-sgRNA-ssODN (SEQ ID NO: 385)for intracellular huANP32A N129I/D130N point mutation and a donorsequence huANP32B-sgRNA-ssODN (SEQ ID NO: 388) for huANP32B N129I/D130Npoint mutation were synthesized. The synthesized single nucleotidesequence was diluted to 10 uM with distilled water for future use.

TABLE 26 primer sequences primer name primer sequence (5′-3′)huANP32A-sgRNA-F, TCTCGGTAGTCGTTCAGGTGTTT SEQ ID NO: 383 TAGAGCTAGAAAThuANP32A-sgRNA-R, ACCTGAACGACTACCGAGACGG SEQ ID NO: 384 TGTTTCGTCCTTTChuANP32A-sgRNA-ssODN, TGAGTTGCGGGAGGAGCTTGAA SEQ ID NO: 385CACATTTTCTCGGTAGTTAATCA GGTTGGTTACCTCGCAATTGAA AAGGTCTAAGCTChuANP32B-sgRNA-F, TCTCGGTAGTCATTCAGGTGTTT SEQ ID NO: 386TAGAGCTAGAAATAGC huANP32B-sgRNA-R, ACCTGAATGACTACCGAGACGG SEQ ID NO: 387TGTTTCGTCCTTTC huANP32B-sgRNA-ssODN, TAAGCTGGGGCAGGAGCTTGAASEQ ID NO: 388 GACACTCTCTCGGTAGTTAATCA GGTTGGTAACCTCACAGTTAAAGAGGTCCAGGCTT

1 ug of eukaryotic plasmid pMJ920 (Addge plasma #42234) expressingCas9-GFP protein, 1 ug of pMD18T-U6-huANP32A-129/130-sgRNA recombinantplasmid and 0.5 μL of diluted huANP32A-sgRNA-ssODN (SEQ ID NO: 385) weremixed with lipofectamine 2000 at a ratio of 1:2.5, and then transfectedinto 293T cells; 1 ug of eukaryotic plasmid pMJ920 (Addge plasma #42234)expressing Cas9-GFP protein, 1 ug of pMD18T-U6-huANP32B-129/130-sgRNArecombinant plasmid and 0.5 μL of diluted huANP32B-sgRNA-ssODN (SEQ IDNO: 388) were mixed with lipofectamine 2000 at a ratio of 1:2.5, andthen transfected into 293T cells. After 48 hours, GFP-positive cellswere screened by an ultra-speed flow cytometry sorting system, andplated in a 96-well plate at a single cell/well for about 10 days;single-cell clones were picked for expansion and culture, and thencellular RNA was extracted according to the procedure using a SimplyPtotal RNA extraction Kit (purchased from Bioflux, cat # BSC52M1), andcDNA was synthesized using a reverse transcription Kit of Takara Co.,Ltd (PrimneScript™ RT reagent Kit with gDNA Eraser (Perfect read Time),Cat.RR047A); and sgRNA-targeting fragments of huANP32A (using primerpair SEQ ID NO:389 and SEQ ID NO: 390) and huANP32B (using primer pairSEQ ID NO:391 and SEQ ID NO: 392) were amplified by KOD Fx Neopolymerase using the cDNA as a template, and the amplification primerswere shown in Table 27, wherein the size of huANP32A amplified fragmentwas 570 bp, and the size of huANP32B amplified fragment was 572 bp.Single-cell clones that were verified to have correct gene mutations bysequencing were subject to western blotting identification andsubsequent experimental studies. The cell lines of mutations of both the129/130 amino acid sites of huANP32A and huANP32B were obtained afterthe first round of obtaining the huANP32B single-knockout cell line,followed by another round of knockout screening, and the transfectionsystem and screening steps were as described above. For the constructedcell lines, the fragments of interest of huANP32A and huANP32B wereamplified; the effect of single mutation and double mutation wereverified; the identification and sequencing results of cell lines wereshown in FIG. 32, wherein FIG. 32A showed the sequencing results ofhuANP32A and huANP32B129/130-site amino acids of the huANP32AN129I/D130N single-mutant cell line (named as A21 IN), FIG. 32B showedthe sequencing results of huANP32A and huANP32B129/130-site amino acidsof the huANP32B N129I/D130N single-mutant cell line (named as B5 IN),and FIG. 32C showed the sequencing results of huANP32A and huANP32B129/130-site amino acids of the double-mutant cell line (named as ABIN).

TABLE 27 the primer sequences for identification ofhuANP32A and huANP32B point-mutant cell line primer nameprimer sequence (5′-3′) hu32A gRNA-F, CCAAAGTTAAACAAACTTAAGAAGCSEQ ID NO: 389 TTGAACTAAGC hu32A gRNA-R, TTAGTCATCATCTTCTCCCTCATCTTCSEQ ID NO: 390 AGGTTCT hu32B gRNA-F, AGCTGCCTAAATTGAAAAAGCTTGAASEQ ID NO: 391 CTC hu32B gRNA-R, TTAATCATCTTCTCCTTCATCATCTGTTSEQ ID NO: 392 TCTCTC

Anti-PHAP1 antibody (purchased from Abcam, cat # ab51013) andAnti-PHAPI2/APRIL antibody [EPR14588] (purchased from Abcam, cat #ab200836) were used in Western bloting; β-actin is used as the internalcontrol gene, and the antibody of Monoclonal Anti-β-Actin antibodyproduced in mouse (purchased from Sigma, cat # A1978-200UL) was used,and the results were shown in FIG. 33.

Based on the above, we successfully constructed a huANP32A N129I/D130Nsingle-mutant cell line (named as A21 IN), a huANP32B N129I/D130Nsingle-mutant cell line (named as B5 IN) and a huANP32A and huANP32Bdouble-mutant cell line (named as AB IN), which were used for subsequentexperiments.

Example 27: Influence of ANP32 Protein on the Replication of InfluenzaVirus

The double-knockout cell line (DKO) constructed in Example 2, the A21 INcell line, B5 IN cell line and AB IN cell line constructed in Example26, and the wild-type 293T cell line were plated in a 12-well plate at3×10⁵/well; after 20 hours, the 5 plasmids of H1N1_(SC09) polymerasereporter system were co-transfected. The transfection system was: PB1plasmid (80 ng), PB2 plasmid (80 ng), PA plasmid (40 ng), NP plasmid(160 ng), pMD18T-vLuc plasmid (80 ng) and pRL-TK plasmid (10 ng), andeach group was provided with triplicate wells.24 h after transfection,the cells were lysed as described in Example 3 and the activity ofpolymerase was detected; the result showed that: the activity ofH1N1_(SC09) polymerase on the single-mutant cell line of A21 IN cellline and B5 IN cell line was slightly different from that of the wildtype 293T cell, and was reduced by only 3-5 times, while the activity ofthe polymerase on the AB IN cell line and the DKO cell line wassignificantly reduced by about 3000-5000 times, and the result was shownin FIG. 34.

The above experiment was repeated with the H7N9_(AH13) polymerasereporter system instead of the H1N1_(SC09) polymerase reporter system;the result showed that: the activity of H7N9_(AH13) polymerase on thesingle-mutant cell line of A21 IN cell line and B5 IN cell line wasslightly different from that of the wild type 293T cell, and was reducedby only about 10 times, while the activity of the H7N9_(AH13) polymeraseon the AB IN cell line and the DKO cell line was significantly reducedby about 7000-10000 times, and the result was shown in FIG. 35.

Example 28: Alignment of Amino Acid Sequences of Avian-Derived ANP32AProteins

The amino acid sequences of avian-derived ANP32A proteins (chANP32A,zfANP32A, dkANP32A and tyANP32A) were compared (see FIG. 36), whereinthe corresponding relationship of amino acid positions 129, 130, 149,151, and positions 60, 63, 87, 90, 93, 95, 112, 115 and 118 between eachavian-derived ANP32A protein and chANP32A protein was shown in Table 28.

TABLE 28 chANP32A 129 130 149 151 60 63 87 90 93 95 112 115 118 zfANP32A129 130 149 151 60 63 87 90 93 95 112 115 118 dkANP32A 119 120 139 14150 53 77 80 83 85 102 105 108 tyANP32A 128 129 148 150 59 62 86 89 92 94111 114 117

As demonstrated in Example 21, the mutation of amino acids in the aminoacid segment 71-180 of chANP32A to alanine resulted in a complete lossof the ability of the protein to support polymerase activity, while themutation of amino acids in the amino acid segments 61-70 and 191-200 toalanine resulted in a decrease of the ability of the protein to supportpolymerase activity.

As can be seen from the alignment of the amino acid sequences of thezfANP32A protein and the chANP32A protein in FIG. 36, the sequences ofthe following amino acid segments of zfANP32A are completely identicalto corresponding amino acid sequences in chANP32A: amino acid segment61-71, amino acid segment 73-75, amino acid segment 77-99, amino acidsegment 101-165, amino acid segment 167-169, amino acid segment 171-180and amino acid segment 181-200, therefore, the mutation of amino acidsin amino acid segment 61-71, amino acid segments 73-75, 77-99, 101-165,167-169 and 171-180 of the zfANP32A protein to alanine also resulted ina complete loss of the ability of the protein to support the polymeraseactivity; the mutation of amino acids in amino acid segments 61-70 and191-200 to alanine resulted in a decrease in the ability to supportpolymerase activity.

From the alignment of the amino acid sequences between the dkANP32Aprotein and the chANP32A protein in FIG. 36, it was found that the aminoacid at position 66 of the dkANP32A protein was I, while the amino acidat position 76 of the chANP32A protein was V, and that the amino acidsat positions 164-167 of dkANP32A were deleted (aligned with positions176-179 of chANP32 protein). It can be seen that the mutation of aminoacids in the amino acid segments 61-65 and 67-166 of the dkANP32Aprotein to alanine resulted in a complete loss of the ability of theprotein to support polymerase activity, while the mutation of aminoacids in the amino acid segments 51-60 and 177-186 to alanine resultedin a decrease of the ability of the protein to support polymeraseactivity.

From the alignment of the amino acid sequences between the tyANP32Aprotein and the chANP32A protein in FIG. 36, it can be seen that theamino acid sequence of the tyANP32A protein is completely identical tothe amino acid sequence of the chANP32A protein. It can be seen that themutation of amino acids in the amino acid segment 60-179 of the tyANP32Aprotein to alanine resulted in a complete loss of the ability of theprotein to support polymerase activity, while the mutation of aminoacids in the amino acid segments 60-69 and 190-199 to alanine resultedin a decrease of the ability of the protein to support polymeraseactivity.

Example 29: Alignment of Amino Acid Sequences Between Avian-DerivedANP32B Protein and huANP32B Protein

The amino acid sequences of avian-derived ANP32B proteins (chANP32B,dkANP32B(XP_012963723.1) and tyANP32B(XP_010723174.1)) were comparedwith the amino acid sequence of huANP32B protein (see FIG. 37), whereinthe corresponding relationship of amino acid positions 129, 130, 149,151, and positions 60, 63, 87, 90, 93, 95, 112, 115 and 118 between eachavian-derived ANP32B protein and huANP32B protein was shown in Table 29.

huANP32B 129 130 149 151 60 63 87 90 93 95 112 115 118 chANP32B 129 130149 151 60 63 87 90 93 95 112 115 118 dkANP32B 143 144 163 165 74 77 101103 107 109 126 129 132 tyANP32B 81 82 101 103 12 15 39 42 45 47 64 6770

As demonstrated in Example 19, the mutation of amino acids in the aminoacid segment 51-160 of huANP32B to alanine resulted in a complete lossof the ability of the protein to support polymerase activity, while themutation of amino acids in the amino acid segments 41-50 and 161-170 toalanine resulted in a decrease of the ability of the protein to supportpolymerase activity.

As can be seen from the alignment of the amino acid sequences of thechANP32B protein and the huANP32B protein in FIG. 37, the sequences ofthe following amino acid segments of chANP32B are completely identicalto corresponding amino acid sequences in huANP32B protein; amino acidsegment 43-48, amino acid segment 50-52, amino acid segment 58-63, aminoacid segment 68-72, amino acid segment 74-76, amino acid segment 78-80,amino acid segment 83-85, amino acid segment 88-99, amino acid segment101-103, amino acid segment 105-112, amino acid segment 117-126, aminoacid segment 131-136, amino acid segment 138-147 and amino acid segment153-159, therefore, the mutation of amino acids in amino acid segments50-52, 58-63, 68-72, 74-76, 78-80, 83-85, 88-99, 101-103, 105-112,117-126, 131-136, 138-147 and 153-159 of the chANP32B protein to alaninealso resulted in a complete loss of the ability of the protein tosupport the polymerase activity; the mutation of amino acids in aminoacid segment 43-48 to alanine resulted in a decrease in the ability tosupport polymerase activity.

As can be seen from the alignment of the amino acid sequences of thedkANP32B protein and the huANP32B protein in FIG. 37, the sequences ofthe following amino acid segments of dkANP32B are completely identicalto corresponding amino acid sequences in huANP32B protein; amino acidsegment 57-62 (corresponding to the amino acid segment 43-48 ofhuANP32B), amino acid segment 70-77 (corresponding to the amino acidsegment 56-63 of huANP32B), amino acid segment 82-86 (corresponding toamino acid segment 68-72 of huANP32B), amino acid segment 88-90(corresponding to the amino acid segment 74-76 of huANP32B), amino acidsegment 92-94 (corresponding to the amino acid segment 78-80 ofhuANP32B), amino acid segment 97-99 (corresponding to the amino acidsegment 83-85 of huANP32B), amino acid segment 102-113 (corresponding tothe amino acid segment 88-99 of huANP32B), amino acid segment 115-117(corresponding to the amino acid segment 101-103 of huANP32B), aminoacid segment 119-126 (corresponding to the amino acid segment 105-112 ofhuANP32B), amino acid segment 131-140 (corresponding to the amino acidsegment 117-126 of huANP32B), amino acid segment 145-150 (correspondingto the amino acid segment 131-136 of huANP32B), amino acid segment152-161 (corresponding to the amino acid segment 138-147 of huANP32B)and amino acid segment 166-173 (corresponding to the amino acid segments152-159 of huANP32B), therefore, the mutation of amino acids in aminoacid segments 70-77, 82-86, 88-90, 92-94, 97-99, 102-113, 115-117,119-126, 131-140, 145-150, 152-161 and 166-173 of the dkANP32B proteinto alanine also resulted in a complete loss of the ability of theprotein to support the polymerase activity; the mutation of amino acidsin amino acid segment 57-62 to alanine resulted in a decrease in theability to support polymerase activity.

As can be seen from the alignment of the amino acid sequences of thetyANP32B protein and the huANP32B protein in FIG. 37, the sequences ofthe following amino acid segments of tyANP32B are completely identicalto corresponding amino acid sequence in huANP32B protein: amino acidsegment 10-15 (corresponding to the amino acid segment 58-63 ofhuANP32B), amino acid segment 20-24 (corresponding to amino acid segment68-72 of huANP32B), amino acid segment 26-28 (corresponding to the aminoacid segment 74-76 of huANP32B), amino acid segment 30-32 (correspondingto the amino acid segment 78-80 of huANP32B), amino acid segment 35-37(corresponding to the amino acid segment 83-85 of huANP32B), amino acidsegment 40-51 (corresponding to the amino acid segment 88-99 ofhuANP32B), amino acid segment 53-55 (corresponding to the amino acidsegment 101-103 of huANP32B), amino acid segment 57-64 (corresponding tothe amino acid segment 105-112 of huANP32B), amino acid segment 69-78(corresponding to the amino acid segment 117-126 of huANP32B), aminoacid segment 83-88 (corresponding to the amino acid segment 131-136 ofhuANP32B), amino acid segment 90-99 (corresponding to the amino acidsegment 138-147 of huANP32B) and amino acid segment 105-111(corresponding to the amino acid segments 153-159 of huANP32B),therefore, the mutation of amino acids in amino acid segments 20-24,26-28, 30-32, 35-37, 40-51, 53-55, 57-64, 69-78, 83-88, 90-99 and105-111 of the tyANP32B protein to alanine also resulted in a completeloss of the ability of the protein to support the polymerase activity.

Example 30: Alignment of Amino Acid Sequences Between Mammalian ANP32AProtein and chANP32A Protein

The amino acid sequences of mammalian ANP32A proteins (huANP32A,dogANP32A (NP_001003013.2), eqANP32A, muANP32A, pgANP32A) were comparedwith the amino acid sequence of chANP32A protein (see FIG. 38), whereinthe corresponding relationship of amino acid positions 129, 130, 149,151, and positions 60, 63, 87, 90, 93, 95, 112, 115 and 118 between eachmammalian ANP32A protein and chANP32A protein was shown in Table 30.

TABLE 30 huANP32A 129 130 149 151 60 63 87 90 93 95 112 115 118dogANP32A 129 130 149 151 60 63 87 90 93 95 112 115 118 eqANP32A 129 130149 151 60 63 87 90 93 95 112 115 118 muANP32A 129 130 149 151 60 63 8790 93 95 112 115 118 pgANP32A 129 130 149 151 60 63 87 90 93 95 112 115118

As demonstrated in Example 21, the mutation of amino acids in the aminoacid segment 71-180 of chANP32A to alanine resulted in a complete lossof the ability of the protein to support polymerase activity, while themutation of amino acids in the amino acid segments 61-70 and 191-200 toalanine resulted in a decrease of the ability of the protein to supportpolymerase activity.

As can be seen from the alignment of the amino acid sequences of thehuANP32A protein and the chANP32A protein in FIG. 38, the sequences ofthe following amino acid segments of huANP32A are completely identicalto corresponding amino acid sequences in chANP32A: amino acid segment41-54, amino acid segment 58-75, amino acid segment 77-102 and aminoacid segment 104-170, therefore, the mutation of amino acids in aminoacid segments 77-102 and 104-170 of the huANP32A protein to alanine alsoresulted in a complete loss of the ability of the protein to support thepolymerase activity; the mutation of amino acids in amino acid segment61-70 to alanine resulted in a decrease in the ability to supportpolymerase activity.

As can be seen from the alignment of the amino acid sequences of theeqANP32A protein and the chANP32A protein in FIG. 38, the sequences ofthe following amino acid segments of eqANP32A are completely identicalto corresponding amino acid sequences in chANP32A: amino acid segment41-54, amino acid segment 56-75, amino acid segment 77-102 and aminoacid segment 104-169, therefore, the mutation of amino acids in aminoacid segments 77-102 and 104-169 of the eqANP32A protein to alanine alsoresulted in a complete loss of the ability of the protein to support thepolymerase activity; the mutation of amino acids in amino acid segment61-70 to alanine resulted in a decrease in the ability to supportpolymerase activity.

As can be seen from the alignment of the amino acid sequences of thedogANP32A protein and the chANP32A protein in FIG. 38, the sequences ofthe following amino acid segments of dogANP32A are completely identicalto corresponding amino acid sequences in chANP32A: amino acid segment41-54, amino acid segment 56-75, amino acid segment 77-102 and aminoacid segment 104-169, therefore, the mutation of amino acids in aminoacid segments 77-102 and 104-169 of the dogANP32A protein to alaninealso resulted in a complete loss of the ability of the protein tosupport the polymerase activity; the mutation of amino acids in aminoacid segment 61-70 to alanine resulted in a decrease in the ability tosupport polymerase activity.

As can be seen from the alignment of the amino acid sequences of thepgANP32A protein and the chANP32A protein in FIG. 38, the sequences ofthe following amino acid segments of pgANP32A are completely identicalto corresponding amino acid sequences in chANP32A: amino acid segment41-54, amino acid segment 56-75, amino acid segment 77-102, amino acidsegment 106-155 and amino acid segment 157-169, therefore, the mutationof amino acids in amino acid segments 77-102,106-155 and 157-169 of thepgANP32A protein to alanine also resulted in a complete loss of theability of the protein to support the polymerase activity; the mutationof amino acids in amino acid segment 61-70 to alanine resulted in adecrease in the ability to support polymerase activity.

As can be seen from the alignment of the amino acid sequences of themuANP32A protein and the chANP32A protein in FIG. 38, the sequences ofthe following amino acid segments of muANP32A are completely identicalto corresponding amino acid sequences in chANP32A: amino acid segment41-54, amino acid segment 59-72, amino acid segment 80-90, amino acidsegment 92-102, amino acid segment 104-129, amino acid segment 131-141,amino acid segment 144-151, amino acid segment 153-159 and amino acidsegment 161-165, therefore, the mutation of amino acids in amino acidsegments 80-90, 92-102, 104-129, 131-141, 144-151, 153-159 and 161-165of the muANP32A protein to alanine also resulted in a complete loss ofthe ability of the protein to support the polymerase activity; themutation of amino acids in amino acid segments 61-70 to alanine resultedin a decrease in the ability to support polymerase activity.

Example 31: Alignment of Amino Acid Sequence of Mammalian ANP32B Protein

The amino acid sequences of mammalian ANP32B proteins (huANP32B,dogANP32B (XP_013973354.1), eqANP32B(XP_023485491.1),muANP32B(NP_570959.1), pgANP32B(XP_020922136.1)) were compared (see FIG.39), wherein the corresponding relationship of amino acid positions 129,130, 149, 151, and positions 60, 63, 87, 90, 93, 95, 112, 115 and 118among various mammalian ANP32B proteins was shown in Table 31.

TABLE 31 huANP32B 129 130 149 151 60 63 87 90 93 95 112 115 118dogANP32B 136 137 156 158 67 70 94 97 100 102 119 122 125 eqANP32B 129130 149 151 60 63 87 90 93 95 112 115 118 muANP32B 129 130 149 151 60 6387 90 93 95 112 115 118 pgANP32B 129 130 149 151 60 63 87 90 93 95 112115 118

As demonstrated in Example 19, the mutation of amino acids in the aminoacid segment 51-160 of huANP32B to alanine resulted in a complete lossof the ability of the protein to support polymerase activity, while themutation of amino acids in the amino acid segments 41-50 and 161-170 toalanine resulted in a decrease of the ability of the protein to supportpolymerase activity.

As can be seen from the alignment of the amino acid sequences of theeqANP32B protein and the huANP32B protein in FIG. 39, the sequences ofthe following amino acid segments of eqANP32B are completely identicalto corresponding amino acid sequences in huANP32B: amino acid segment41-72, amino acid segment 74-112 and amino acid segment 115-170,therefore, the mutation of amino acids in amino acid segments 51-72,74-112 and 115-160 of the eqANP32B protein to alanine also resulted in acomplete loss of the ability of the protein to support the polymeraseactivity; the mutation of amino acids in amino acid segments 41-50 and161-170 to alanine resulted in a decrease in the ability to supportpolymerase activity.

As can be seen from the alignment of the amino acid sequences of thepgANP32B protein and the huANP32B protein in FIG. 39, the sequences ofthe following amino acid segments of pgANP32B are completely identicalto corresponding amino acid sequences in huANP32B: amino acid segment41-72, amino acid segment 78-142, amino acid segment 144-150 and aminoacid segment 154-169, therefore, the mutation of amino acids in aminoacid segments 51-72, 78-142, 144-150 and 154-160 of the pgANP32B proteinto alanine also resulted in a complete loss of the ability of theprotein to support the polymerase activity; the mutation of amino acidsin amino acid segments 41-50 and 161-169 to alanine resulted in adecrease in the ability to support polymerase activity.

As can be seen from the alignment of the amino acid sequences of themuANP32B protein and the huANP32B protein in FIG. 39, the sequences ofthe following amino acid segments of muANP32B are completely identicalto corresponding amino acid sequences in huANP32B: amino acid segment41-50, amino acid segment 60-81, amino acid segment 90-98, amino acidsegment 100-110, amino acid segment 114-121, amino acid segment 123-127,amino acid segment 130-133, amino acid segment 138-142 and amino acidsegment 144-159, therefore, the mutation of amino acids in amino acidsegments 60-81, 90-98, 100-110, 114-121, 123-127, 130-133, 138-142 and144-159 of the muANP32B protein to alanine also resulted in a completeloss of the ability of the protein to support the polymerase activity;the mutation of amino acids in amino acid segment 41-50 to alanineresulted in a decrease in the ability to support polymerase activity.

As can be seen from the alignment of the amino acid sequences of thedogANP32B protein and the huANP32B protein in FIG. 39, the sequences ofthe following amino acid segments of dogANP32B are completely identicalto corresponding amino acid sequences in huANP32B: amino acid segment48-79 (corresponding to the amino acid segment 41-72 of the huANP32Bprotein), amino acid segment 81-120 (corresponding to the amino acidsegment 74-113 of the huANP32B protein), amino acid segment 122-159(corresponding to the amino acid segment 115-152 of the huANP32Bprotein), amino acid segment 161-177 (corresponding to amino acidsegment 154-170 of the huANP32B protein), therefore, the mutation ofamino acids in amino acid segments 58-79, 81-120, 122-159 and 161-167 ofthe dogANP32B protein to alanine also resulted in a complete loss of theability of the protein to support the polymerase activity; the mutationof amino acids in amino acid segments 48-57 and 168-177 to alanineresulted in a decrease in the ability to support polymerase activity.

Example 32: Construction of Murine ANP32B Protein Expression Vector andMutation Vector

The nucleotide sequence of murine ANP32B (muANP32B) was shown in GenBankNo. NM_130889.3, the amino acid sequence thereof was shown in GenBankNo. NP_570959.1; the PCAGGS-muANP32B recombinant plasmid was constructedaccording to the construction method described in Example 1, and theprotein expression was detected by Western blotting as the detectionmethod described in Example 1, and the result was shown in FIG. 40.

According to the screening results of huANP32B point mutation describedin Example 8, muANP32B was subjected to a point mutant construction ofS129I/D130N (using primer pair of SEQ ID NO: 393 and SEQ ID NO: 394)(see Table 32 for primers, and mutated bases were underlined) by usingoverlapping PCR with the PCAGGS-muANP32B recombinant plasmid as atemplate, wherein the process was as described for the construction of apoint mutant in Example 8. As described above, the obtained plasmid wasnamed as PCAGGS-muANP32B S129I/D130N. After verification by sequencing,the plasmids were extracted in large amount for further transfection.

TABLE 32 Primers for point mutation of S129I/D130N on muANP32Bprimer name primer sequence (5′-3′) muB_S129I/D130N_FCTAACCGGATTAACTACCGAGAAACTGTCTT SEQ ID NO: 393 muB_S129I/D130N_RTCGGTAGTTAATCCGGTTAGTGACCTCACA SEQ ID NO: 394

Example 33: Influence of Murine ANP32B Protein Mutant on PolymeraseActivity

Double-knockout cell line (DKO) was plated in a 12-well plate at3×10⁵/well and after 20 h was co-transfected with the H7N9_(AH13)polymerase reporter system according to the transfection systemdescribed in Example 8, and the result showed that: muANP32B S129I/D130Ncompletely lost its support for H7N9_(AH13) polymerase activity ascompared with muANP32B, as shown in FIG. 41.

1. A mutated ANP32 protein having one or more mutations selected from: the amino acid at position 129 substituted with isoleucine I, lysine K, aspartic acid D, valine V, proline P, tryptophan W, histidine H, arginine R, glutamine Q, glycine G, or glutamic acid E, the amino acid at position 130 substituted with asparagine N, phenylalanine F, lysine K, leucine L, valine V, proline P, isoleucine I, methionine M, tryptophan W, histidine H, arginine R, glutamine Q, or tyrosine Y, the amino acid at position 149 substituted with alanine A, the amino acid at position 151 substituted with alanine A, or the amino acids at positions 60 and 63, positions 87, 90, 93 and 95, positions 112, 115 and 118 are substituted with alanine, wherein: when the ANP32 protein is chicken ANP32B protein, duck ANP32B protein or turkey ANP32B protein, the amino acid at position 129 is not isoleucine I and the amino acid at position 130 is not asparagine N, when the ANP32 protein is murine ANP32A, the amino acid at position 130 is not alanine A, wherein, when the ANP32 protein is an ANP32A protein, the amino acid positions correspond to the amino acid positions of a chicken ANP32A protein of GenBank No. XP_413932.3; when the ANP32 protein is an ANP32B protein, the amino acid positions correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1, and wherein the ANP32 protein is selected from ANP32A or ANP32B derived from different species. 2-27. (canceled)
 28. A mutated ANP32 protein, wherein one or more of the the following amino acid segments are deleted or substituted with alanine: amino acids at positions 61-70, amino acids at positions 71-80, amino acids at positions 81-90, amino acids at positions 91-100, amino acids at positions 101-110, amino acids at positions 111-120, amino acids at positions 121-130, amino acids at positions 131-140, amino acids at positions 141-150, or amino acids at positions 151-160, wherein, when the ANP32 protein is an ANP32A protein, the amino acid positions correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3; when the ANP32 protein is an ANP32B protein, the amino acid positions correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1, wherein the ANP32 protein is selected from ANP32A or ANP32B derived from different species.
 29. A mutated ANP32 protein, wherein when the ANP32 protein is human ANP32B protein, one or more of the following amino acid segments are deleted or substituted with alanine: amino acids at positions 21-30, amino acids at positions 41-50, amino acids at positions 51-60 or amino acids at positions 161-170, or the amino acid positions, which correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1.
 30. A mutated ANP32 protein, wherein when the ANP32 protein is chicken ANP32B protein, one or more of the following amino acid segments are deleted or substituted with alanine: amino acids at positions 161-170, amino acids at positions 171-180 or amino acids at positions 191-200, or the amino acid positions, which correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3.
 31. A method of reducing the polymerase activity of an influenza virus, comprising mutating the ANP32 protein to have one or more of the following mutations: the amino acid at position 129 is substituted with isoleucine I, lysine K, aspartic acid D, valine V, proline P, tryptophan W, histidine H, arginine R, glutamine Q, glycine G, or glutamic acid E, the amino acid at position 130 is substituted with asparagine N, phenylalanine F, lysine K, leucine L, valine V, proline P, isoleucine I, methionine M, tryptophan W, histidine H, arginine R, glutamine Q, or tyrosine Y, the amino acid at position 149 is substituted with alanine A, the amino acid at position 151 is substituted with alanine A, or the amino acids at positions 60 and 63, positions 87, 90, 93 and 95, positions 112, 115 and 118 are substituted with alanine, wherein: when the ANP32 protein is a chicken ANP32B protein, duck ANP32B protein or turkey ANP32B protein, the amino acid at position 129 is not isoleucine and the amino acid at position 130 is not asparagine N, when the ANP32 protein is murine ANP32A, the amino acid at position 130 is not alanine A, wherein, when the ANP32 protein is an ANP32A protein, the amino acid positions correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3; when the ANP32 protein is an ANP32B protein, the amino acid positions correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1, and wherein the ANP32 protein is selected from ANP32A or ANP32B derived from different species.
 32. The method of claim 31, wherein the polymerase activity of influenza virus is lost, wherein the ANP32 protein is subjected to one or more mutations selected from the following: the amino acid at position 129 is substituted with isoleucine I, lysine K or aspartic acid D, the amino acid at position 130 is substituted with asparagine N, phenylalanine F or lysine K, or the amino acids at positions 87, 90, 93 and 95, positions 112, 115 and 118 are substituted with alanine.
 33. A method of reducing the polymerase activity of influenza virus, comprising deleting or substituting one or more of the amino acid segments of ANP32 protein with alanine, wherein the one or more of the amino acid segments are selected from the following: amino acids at positions 61-70, amino acids at positions 71-80, amino acids at positions 81-90, amino acids at positions 91-100, amino acids at positions 101-110, amino acids at positions 111-120, amino acids at positions 121-130, amino acids at positions 131-140, amino acids at positions 141-150, or amino acids at positions 151-160, wherein, when the ANP32 protein is an ANP32A protein, the amino acid positions correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3; when the ANP32 protein is an ANP32B protein, the amino acid positions correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1, and wherein the ANP32 protein is selected from ANP32A or ANP32B derived from different species.
 34. The method of claim 33, wherein the polymerase activity of influenza virus is lost, wherein one or more of the amino acid segments of ANP32 protein are deleted or substituted with alanine, wherein the amino acid segment is selected from the following: amino acids at positions 71-80, amino acids at positions 81-90, amino acids at positions 91-100, amino acids at positions 101-110, amino acids at positions 111-120, amino acids at positions 121-130, amino acids at positions 131-140, amino acids at positions 141-150, or amino acids at positions 151-160.
 35. A method of reducing the polymerase activity of influenza virus, comprising deleting or substituting one or more of the amino acid segments of human ANP32B protein with alanine, wherein the one or more of the amino acid segments are selected from the following: amino acids at positions 21-30, amino acids at positions 41-50, amino acids at positions 51-60 or amino acids at positions 161-170, or the amino acid positions, which correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1, wherein the ANP32 protein is selected from ANP32A or ANP32B derived from different species.
 36. A method of reducing the polymerase activity of influenza virus, comprising deleting or substituting one or more of the amino acid segments of chicken ANP32A protein with alanine, wherein the one or more of the amino acid segments are selected from the following: amino acids at positions 161-170, amino acids at positions 171-180, amino acids at positions 191-200, or the amino acid positions, which correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3, wherein the ANP32 protein is selected from ANP32A or ANP32B derived from different species.
 37. The method of claim 36, wherein the polymerase activity of influenza virus is lost, wherein, one or more of the amino acid segments of chicken ANP32A protein are deleted or substituted with alanine, wherein the one or more of the amino acid segments are selected from amino acids at positions 161-170, or amino acids at positions 171-180.
 38. A kit comprising at least one reagent or a set of reagents, wherein the at least one reagent or a set of reagents is used to determine the type of amino acid at one or more positions of an ANP32 protein selected from the following: amino acid at position 129, amino acid at position 130, amino acid at position 149, amino acid at position 151, amino acid at position 60, amino acid at position 63, amino acid at position 87, amino acid at position 90, amino acid at position 93, amino acid at position 95, amino acid at position 112, amino acid at position 115, or amino acid at position 118, wherein when the amino acid at position 129 is isoleucine I, lysine K, aspartic acid D, valine V, proline P, tryptophan W, histidine H, arginine R, glutamine Q, glycine G, or glutamic acid E, when the amino acid at position 130 is asparagine N, phenylalanine F, lysine K, leucine L, valine V, proline P, isoleucine I, methionine M, tryptophan W, histidine H, arginine R, glutamine Q, or tyrosine Y, when the amino acid at position 149 is alanine A, when the amino acid at position 151 is alanine A, when the amino acids at positions 60 and 63, at positions 87, 90, 93 and 95, at positions 112, 115, and 118 are alanine, the ability of the ANP32 protein to support the activity of influenza polymerase is decreased, wherein, when the ANP32 protein is an ANP32A protein, the amino acid positions correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3; when the ANP32 protein is an ANP32B protein, the amino acid positions correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1.
 39. An oligonucleotide primer for determining the type of amino acid of ANP32 protein selected from the following: amino acid at position 129, amino acid at position 130, amino acid at position 149, amino acid at position 151, amino acid at position 60, amino acid at position 63, amino acid at position 87, amino acid at position 90, amino acid at position 93, amino acid at position 95, amino acid at position 112, amino acid at position 115, or amino acid at position 118, wherein: when the amino acid at position 129 is isoleucine I, lysine K, aspartic acid D, valine V, proline P, tryptophan W, histidine H, arginine R, glutamine Q, glycine G, or glutamic acid E, when the amino acid at position 130 is asparagine N, phenylalanine F, lysine K, leucine L, valine V, proline P, isoleucine I, methionine M, tryptophan W, histidine H, arginine R, glutamine Q, or tyrosine Y, when the amino acid at position 149 is alanine A, when the amino acid at position 151 is alanine A, when the amino acids at positions 60, 63, or the amino acids at positions 87, 90, 93 and 95, or the amino acids at positions 112, 115 and 118 are alanine, the ability of ANP32 protein to support the activity of influenza polymerase is decreased, wherein, when the ANP32 protein is an ANP32A protein, the amino acid positions correspond to the amino acid positions of the chicken ANP32A protein of GenBank No. XP_413932.3; when the ANP32 protein is an ANP32B protein, the amino acid positions correspond to the amino acid positions of the human ANP32B protein of GenBank No. NP_006392.1, wherein the amino acids at positions 87, 90, 93 and 95 are from a mammalian ANP32B protein, wherein the oligonucleotide primer is preferably at least 20 bases in length and, wherein the oligonucleotide primer is selected from SEQ ID NOs: 155-156, 163-166, 167-256, or 375-380.
 40. A method of screening for a candidate drug for treating an influenza virus infection, comprising: (1) knocking out the ANP32A and/or ANP32B protein from a cell line containing ANP32A and/or ANP32B protein, to obtain a cell line in which ANP32A protein and/or ANP32B protein are knocked out, (2) transfecting the knockout cell line obtained in step (1) with a plasmid encoding ANP32A and/or ANP32B protein and a plasmid encoding influenza virus polymerase, (3) contacting the knockout cell line with a candidate, wherein the contacting can be performed simultaneously with or separately from the transfection of step (2), wherein the cell line treated in step (3) does not express influenza virus polymerase or has reduced expression of influenza virus polymerase compared to a cell line containing ANP32A and/or ANP32B, indicating that the candidate is a candidate drug for treating influenza virus infection, wherein the ANP32A and/or ANP32B protein is derived from chicken, human, zebra finch, duck, turkey, pig, mouse or horse; preferably, the influenza virus is selected from human, canine, avian or equine influenza virus. 